Therapeutic nanoparticles and methods of use thereof

ABSTRACT

Provided herein are therapeutic nanoparticles having a diameter of between 10 nm to 30 nm, and containing a polymer coating, and a nucleic acid containing a sequence complementary to a sequence within a micro-RNA identified as having a role in cancer cell metastasis or anti-apoptotic activity in a cancer cell (e.g., miR-10b) or a sequence within an mRNA encoding a pro-apoptotic protein that is covalently linked to the nanoparticle. Also provided are pharmaceutical compositions containing these therapeutic nanoparticles. Also provided herein are methods of decreasing cancer cell invasion or metastasis in a subject having a cancer and methods of treating a metastatic cancer in a lymph node in a subject that require the administration of these therapeutic nanoparticles to a subject.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 14/233,215, filedApr. 11, 2014, now U.S. Pat. No. 9,763,891, which is a National Stageapplication under 35 U.S.C. § 371 that claims the benefit ofPCT/US2012/047366, filed Jul. 19, 2012, which claims the benefit of U.S.Provisional Application No. 61/510,563 filed Jul. 22, 2011, thedisclosures of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention relates to therapeutic nanoparticples containing apolymer coating and a covalently-linked inhibitory nucleic acid,compositions containing these therapeutic nanoparticles, and methods ofusing these therapeutic nanoparticles.

BACKGROUND

Breast cancer is the second leading cause of cancer-related deaths inwomen of the Western world. In the U.S.A. alone, over 180,000 new casesof breast cancer are diagnosed each year. Of these patients,approximately 25% will die despite aggressive diagnostic and therapeuticintervention. Despite the considerable and recent improvement in breastcancer diagnostics and therapy, the morbidity and mortality associatedwith this disease remain alarming.

The best chance for survival of breast cancer is to detect the cancerbefore it has had a chance to metastasize. Unfortunately, breast cancercan reappear and metastasis can occur even if the cancer was confined tothe breast at the time of detection. Once metastatic breast cancer hasbeen diagnosed, it can be treated. However, in most cases, none of thetreatments lead to long-term survival.

One of the pathways for breast cancer systemic metastasis involves thepassage of cancer cells through the lymphatic system. There isconvincing experimental support for a metastatic breast cancer modelthat has an early state, in which the breast cancer cells are confinedto the breast and the regional lymph nodes, and have not yetmetastasized to distant sites. Additional recent preclinical andclinical immunohistochemical, physiologic, and pathophysiologicmetastatic breast cancer studies support a model of breast cancermetastasis where the breast cancer cells first invade peritumorallymphatics, then spread to locoregional lymph nodes, prior tohematogenous metastasis in a subject.

SUMMARY

This disclosure describes therapeutic nanoparticles that have a diameterof between 2 nm to 200 nm (e.g., between 10 nm and 200 nm, between 2 nmand 30 nm, between 5 nm and 30 nm, between 10 nm and 30 nm, between 15to 25 nm, or between 20 to 25 nm), and contain a polymer coating and anucleic acid containing a sequence that is complementary to a contiguoussequence present within a microRNA (miRNA) identified as having a rolein cancer cell metastasis or invasion, or anti-apoptotic activity in acancer cell (e.g., miR-10b), or a contiguous sequence present within anmRNA encoding a pro-apoptotic protein that is covalently linked to thenanoparticle. In some implementations, the therapeutic nanoparticles aremagnetic (e.g., contain a magnetic core material). Also provided arepharmaceutical compositions containing these therapeutic nanoparticles,and methods of decreasing cancer cell invasion or metastasis in asubject and methods of treating metastatic cancer in a lymph node in asubject that include the administration of these therapeuticnanoparticles.

The therapeutic nanoparticles can have a diameter of between 10 nm to 30nm (e.g., between 15 to 25 nm or between 20 to 25 nm), and can include apolymer coating (e.g., a polymer coating containing dextran) and anucleic acid containing at least 10 (e.g., between 10-15 nucleotides orbetween 15 to 20 nucleotides) contiguous nucleotides within the sequenceof GTGTAACACGTCTATACGCCCA (SEQ ID NO: 17), ATGGGACATCTTGGCTTAAACAC (SEQID NO: 1) or TGTCTAAGCTAAGAT CCCCTTA (SEQ ID NO: 2) that is covalentlylinked to the nanoparticle. In some implementations, the nucleic acidcontains at least one (e.g., at least two, three, or four) modifiednucleotide(s) (e.g., a nucleotide containing a base modification or aribose or deoxyribose modification) or one or more modifications in thephosphate (phosphodiester) backbone. In some implementations, themodified nucleotide is a locked nucleotide. In some embodiments, thenucleic acid is single-stranded or double stranded. In some embodiments,the nucleic acid is a small interfering RNA (siRNA) molecule. In someimplementations, the nucleic acid is covalently-linked to thenanoparticle through a chemical moiety containing a disulfide bond. Insome embodiments, the nucleic acid is covalently linked to thenanoparticle through a chemical moiety containing a thioether bond.

In some implementations, the therapeutic nanoparticle further contains acovalently-linked fluorophore (e.g., a fluorophore that absorbsnear-infrared light). In some embodiments, the fluorophore is covalentlylinked to the nanoparticle through a chemical moiety that contains asecondary amine.

In some embodiments, the therapeutic nanoparticle further contains acovalently-linked targeting peptide. In some embodiments, the targetingpeptide contains an RGD peptide, an EPPT peptide, NYLHNHPYGTVG (SEQ IDNO: 11), SNPFSKPYGLTV (SEQ ID NO: 12), GLHESTFTQRRL (SEQ ID NO: 13),YPHYSLPGSSTL (SEQ ID NO: 14), SSLEPWHRTTSR (SEQ ID NO: 15), LPLALPRHNASV(SEQ ID NO: 16), or βAla-(Arg)₇-Cys (SEQ ID NO: 19). In someembodiments, the targeting peptide is covalently linked to thenanoparticle through a chemical moiety that contains a disulfide bond.In some embodiments, the therapeutic nanoparticle is magnetic.

Also provided are pharmaceutical compositions containing any of themagnetic particles described herein.

Also provided are methods for decreasing cancer cell invasion ormetastasis in a subject having a cancer (e.g., breast cancer) thatinclude administering a therapeutic nanoparticle (any of the therapeuticnanoparticles described herein) to a subject having a cancer, where thetherapeutic nanoparticle is administered in an amount sufficient todecrease cancer cell invasion or metastasis in the subject. In someembodiments, the cancer cell metastasis is from a primary tumor to alymph node in the subject or is from a lymph node to a secondary tissuein a subject. In some embodiments, the cancer cell is selected from thegroup of: a breast cancer cell, a colon cancer cell, a kidney cancercell, a lung cancer cell, a skin cancer cell, an ovarian cancer cell, apancreatic cancer cell, a prostate cancer cell, a rectal cancer cell, astomach cancer cell, a thyroid cancer cell, and a uterine cancer cell.Some embodiments of these methods further include imaging a tissue ofthe subject to determine the location or number of cancer cells in thesubject, or the location of the therapeutic nanoparticles (e.g., thelocation of therapeutic magnetic nanoparticles or therapeuticnanoparticles containing a covalently-linked fluorophore) in thesubject.

In another aspect, the disclosure describes methods of treating ametastatic cancer in a lymph node in a subject. These methods includeadministering a therapeutic nanoparticle (any of the therapeuticnanoparticles described herein) to a lymph node of a subject having ametastatic cancer, where the therapeutic nanoparticle is administered inan amount sufficient to treat a metastastic cancer in a lymph node inthe subject. In some embodiments, the metastatic cancer results from aprimary breast cancer. In some embodiments, the administering results ina decrease (e.g., a significant, detectable, or observable decrease) orstabilization of metastatic tumor size or a decrease (e.g., asignificant, detectable, or observable decrease) in the rate ofmetastatic tumor growth in a lymph node in the subject.

In any of the methods described herein, the therapeutic nanoparticlescan be administered in multiple doses to the subject. In someembodiments of the methods described herein, the therapeuticnanoparticles are administered to the subject at least once a week. Insome embodiments of the methods described herein, the therapeuticnanoparticles are administered to the subject by intravenous,subcutaneous, intraarterial, intramuscular, or intraperitonealadministration. In some embodiments of the methods described herein, thesubject is further administered a chemotherapeutic agent.

The term “magnetic” is used to describe a composition that is responsiveto a magnetic field. Non-limiting examples of magnetic compositions(e.g., any of the therapeutic nanoparticles described herein) cancontain a material that is paramagnetic, superparamagnetic,ferromagnetic, or diamagnetic. Non-limiting examples of magneticcompositions contain a metal oxide selected from the group of:magnetite; ferrites (e.g., ferrites of manganese, cobalt, and nickel);Fe(II) oxides; and hematite, and metal alloys thereof. Additionalmagnetic materials are described herein and are known in the art.

The term “diamagnetic” is used to describe a composition that has arelative magnetic permeability that is less than or equal to 1 and thatis repelled by a magnetic field.

The term “paramagnetic” is used to describe a composition that developsa magnetic moment only in the presence of an externally-applied magneticfield.

The term “ferromagnetic” or “ferromagnetic” is used to describe acomposition that is strongly susceptible to magnetic fields and iscapable of retaining magnetic properties (a magnetic moment) after anexternally-applied magnetic field has been removed.

By the term “nanoparticle” is meant an object that has a diameterbetween about 2 nm to about 200 nm (e.g., between 10 nm and 200 nm,between 2 nm and 100 nm, between 2 nm and 40 nm, between 2 nm and 30 nm,between 2 nm and 20 nm, between 2 nm and 15 nm, between 100 nm and 200nm, and between 150 nm and 200 nm). Non-limiting examples ofnanoparticles include the therapeutic nanoparticles described herein.

By the term “magnetic nanoparticle” is meant a nanoparticle (e.g., anyof the therapeutic nanoparticles described herein) that is magnetic (asdefined herein). Non-limiting examples of magnetic nanoparticles aredescribed herein. Additional magnetic nanoparticles are known in theart.

By the term “polymer coating” is meant at least one molecular layer(e.g., homogenous or non-homogenous) of at least one polymer (e.g.,dextran) applied to a surface of a three-dimensional object (e.g., athree-dimensional object containing a magnetic material, such as a metaloxide). Non-limiting examples of polymers that can be used to generate apolymer coating are described herein. Additional examples of polymersthat can be used to generate a polymer coating are known in the art.Methods for applying a polymer coating to an object (e.g., athree-dimensional object containing a magnetic material) are describedherein and are also known in the art.

By the term “nucleic acid” is meant any single- or double-strandedpolynucleotide (e.g., DNA or RNA, cDNA, semi-synthetic, or syntheticorigin). The term nucleic acid includes oligonucleotides containing atleast one modified nucleotide (e.g., containing a modification in thebase and/or a modification in the sugar) and/or a modification in thephosphodiester bond linking two nucleotides. In some embodiments, thenucleic acid can contain at least one locked nucleotide (LNA).Non-limiting examples of nucleic acids are described herein. Additionalexamples of nucleic acids are known in the art.

By the term “modified nucleotide” is meant a DNA or RNA nucleotide thatcontains at least one modification in its base and/or at least onemodification in its sugar (ribose or deoxyribose). A modified nucleotidecan also contain modification in an atom that forms a phosphodiesterbond between two adjoining nucleotides in a nucleic acid sequence.

By the term “fluorophore” is meant a molecule that absorbs light at afirst wavelength and emits light at a second wavelength, where the firstwavelength is shorter (higher energy) than the second wavelength. Insome embodiments, the first wavelength absorbed by the fluorophore canbe in the near-infrared range. Non-limiting examples of fluorophores aredescribed herein. Additional examples of fluorophores are known in theart.

By the term “near-infrared light” is meant light with a wavelength ofbetween about 600 nm to about 3,000 nm.

By the term “targeting peptide” is meant a peptide that is bound by amolecule (e.g., protein, sugar, or lipid, or combination thereof)present in or on the plasma membrane of a target cell (e.g., a cancercell). As described herein, a targeting peptide can be covalently linkedto a secondary molecule or composition (e.g., any of the therapeuticnanoparticles described herein) to target the secondary molecule orcomposition to a target cell (e.g., a cancer cell). In some embodiments,a targeting peptide that is covalently linked to a secondary molecule orcomposition (e.g., any of the therapeutic nanoparticles describedherein) results in the uptake of the secondary molecule or compositionby the targeted cell (e.g., cellular uptake by endocytosis orpinocytosis). Non-limiting examples of targeting peptides are describedherein. Additional examples of targeting peptides are known in the art.

By the term “small interfering RNA” or “siRNA” is meant adouble-stranded nucleic acid molecule that is capable of mediating RNAinterference in a cell. The process of RNA interference is described inEbalshir et al. (Nature 411:494-498, 2001). Each strand of a siRNA canbe between 19 and 23 nucleotides in length. As used herein, siRNAmolecules need not be limited to those molecules containing only nativeor endogenous RNA nucleotides, but can further encompasschemically-modified nucleotides. Non-limiting examples of siRNA aredescribed herein. Additional examples of siRNA are known in the art.

By the phrase “cancer cell invasion” is meant the migration of a cancercell into a non-cancerous tissue in a subject. Non-limiting examples ofcancer cell invasion include: the migration of a cancer cell into alymph node, the lymph, the vasculature (e.g., adventita, media, orintima of a blood vessel), or an epithelial or endothelial tissue.Exemplary methods for detecting and determining cancer cell invasion aredescribed herein. Additional methods for detecting and determiningcancer cell invasion are known in the art.

By the term “metastasis” is meant the migration of a cancer cell presentin a primary tumor to a secondary, non-adjacent tissue in a subject.Non-limiting examples of metastasis include: metastasis from a primarytumor to a lymph node (e.g., a regional lymph node), bone tissue, lungtissue, liver tissue, and/or brain tissue. The term metastasis alsoincludes the migration of a metastatic cancer cell found in a lymph nodeto a secondary tissue (e.g., bone tissue, liver tissue, or braintissue). In some non-limiting embodiments, the cancer cell present in aprimary tumor is a breast cancer cell, a colon cancer cell, a kidneycancer cell, a lung cancer cell, a skin cancer cell, an ovarian cancercell, a pancreatic cancer cell, a prostate cancer cell, a rectal cancercell, a stomach cancer cell, a thyroid cancer cell, or a uterine cancercell. Additional aspects and examples of metastasis are known in the artor described herein.

By the term “primary tumor” is meant a tumor present at the anatomicalsite where tumor progression began and proceeded to yield a cancerousmass. In some embodiments, a physician may not be able to clearlyidentify the site of the primary tumor in a subject.

By the term “metastatic tumor” is meant a tumor in a subject thatoriginated from a tumor cell that metastasized from a primary tumor inthe subject. In some embodiments, a physician may not be able to clearlyidentify the site of the primary tumor in a subject.

By the term “lymph node” is meant a small spherical or oval-shaped organof the immune system that contains a variety of cells includingB-lymphocytes, T-lymphocytes, and macrophages, which is connected to thelymphatic system by lymph vessels. A variety of lymph nodes are presentin a mammal including, but not limited to: axillary lymph nodes (e.g.,lateral glands, anterior or pectoral glands, posterior or subscapularglands, central or intermediate glands, or medial or subclavicularglands), sentinel lymph nodes, sub-mandibular lymph nodes, anteriorcervical lymph nodes, posterior cervical lymph nodes, supraclavicularlymph nodes, sub-mental lymph nodes, femoral lymph nodes, mesentericlymph nodes, mediastinal lymph nodes, inguinal lymph nodes, subsegmental lymph nodes, segmental lymph nodes, lobar lymph nodes,interlobar lymph nodes, hilar lymph nodes, supratrochlear glands,deltoideopectoral glands, superficial inguinal lymph nodes, deepinguinal lymph nodes, brachial lymph nodes, and popliteal lymph nodes.

By the term “imaging” is meant the visualization of at least one tissueof a subject using a biophysical technique (e.g., electromagentic energyabsorption and/or emission). Non-limiting embodiments of imaginginclude: magnetic resonance imaging (MM), X-ray computed tomography, andoptical imaging.

By the phrase “stabilization of metastatic tumor size” is meant that atumor has reached a stage in which there is only an insignificant ornon-detectable change in the total or approximate volume of a metastatictumor in a subject over time.

By the phrase “rate of metastatic tumor growth” is meant a change in thetotal or approximate volume of a metastatic tumor or a change in thetotal or approximate number of cells present in a metastatic tumor overtime in a subject. The rate of metastatic tumor growth can be determinedusing the exemplary methods described herein. Additional methods fordetermining the rate of metastatic tumor growth are known in the art.

By the term “chemotherapeutic agent” is meant a molecule that can beused to reduce the rate of cancer cell growth or to induce or mediatethe death (e.g., necrosis or apoptosis) of cancer cells in a subject(e.g., a human). In non-limiting examples, a chemotherapeutic agent canbe a small molecule, a protein (e.g., an antibody, an antigen-bindingfragment of an antibody, or a derivative or conjugate thereof), anucleic acid, or any combination thereof. Non-limiting examples ofchemotherapeutic agents include: cyclophosphamide, mechlorethamine,chlorabucil, melphalan, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide,teniposide, tafluposide, azacitidine, axathioprine, capecitabine,cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine,methotrexate, tioguanine, bleomycin, carboplatin, cisplatin,oxaliplatin, all-trans retinoic acid, vinblastine, vincristine,vindesine, vinorelbine, and bevacizumab (or an antigen-binding fragmentthereof). Additional examples of chemotherapeutic agents are known inthe art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing an exemplary therapeutic magneticnanoparticle (MN-RGD-anti-miR-10b) having a dextran coating (light gray)and a covalently-attached tumor-targeting peptide (cRGD), fluorophore(Cy5.5), and a knock-down LNA oligonucleotide targeting humananti-miR-10b (anti-miR10b).

FIG. 2 is a graph of flow cytometry data of MDA-MB-231 human breastcancer cells following a 48-hour incubation with MN-RGD-anti-miR-10b.

FIG. 3 is a graph showing the levels of miR-10b expression determined byquantitative reverse transcriptase polymerase chain reaction (qRT-PCR)in MDA-MB-231 human breast cancer cells following a 48-hour incubationwith MN-RGD-anti-miR-10b or a corresponding magnetic nanoparticle(MN-scr-miR) containing a scrambled nucleic acid rather than theanti-miR10b nucleic acid. The data shown are represented asmean±standard deviation (p<0.0001, n=3).

FIGS. 4A-F are a set of six photomicrographs showing the migration ofMDA-MB-231 cells following treatment with MN-RGD-anti-miR-10b (4E and4F), control magnetic particles (MN-scr-miR; 4C and 4D), or phosphatebuffered saline (PBS)(4A and 4B) in the presence or absence of 10% FBS.

FIGS. 5A-F is a set of six photomicrographs showing the invasion ofMDA-MB-231 cells following treatment with MN-RGD-anti-miR-10b (5E and5F), control magnetic particles (MN-scr-miR; 5C and 5D), or PBS (5A and5B) in the presence or absence of 10% FBS.

FIGS. 6A and 6B are two T2 weighted magnetic resonance images showingthe MN-anti-miR10b accumulation in orthotopic MDA-MB-231-luc-D3H2LNtumors before (6A) and 24-hours after (6B) MN-anti-miR10b injection.Short T2 relaxation times are shown using darker shading, while longerT2 relaxation times are shown in lighter shading.

FIG. 7 is a graph showing the difference in T2 relaxation times (ΔR2) inmsec⁻¹ of orthotopic MDA-MB-231-D3H2LN tumors before or afterMN-anti-miR10b injection (1/T2 pre-injection−1/T2 post-injection, ms).The data shown are the mean±standard deviation (p≤0.01, n=6).

FIGS. 8A, 8B, and 8C are a series of a schematic showing the position ofthe primary tumor and several lymph nodes in the orthotopicMDA-MB-231-luc-D3H2LN mouse model (8A), and two near-infrared opticalimages showing the MN-anti-miR10b accumulation in orthotopicMDA-MB-231-luc-D3H2LN tumors before (8B) and 24-hours after (8C)MN-anti-miR10b administration.

FIGS. 9A-E are a set of five ex vivo images of a excised primary humanbreast cancer tumor (PT)(9A), brachial lymph nodes (BLNs)(9B), aninguinal lymph node (ILN)(9C), a cervical lymph node (CLN)(9D), andmuscle (9E) showing the distribution of MN-RGD-anti-miR-10b (derivedfrom a 23-nm MN precursor) after in vivo delivery into aMDA-MB-231-luc-D3H2LN mouse model.

FIG. 10 is a graph showing the average radiance of excised primary humanbreast cancer tumors (PT), brachial lymph nodes (BLNs), inguinal lymphnodes (ILNs), cervical lymph nodes (CLNs), and muscle following in vivodelivery of MN-RGD-anti-miR-10b. The data shown are the mean±standarddeviation (p≤0.0001, n=6).

FIGS. 11A and 11B are two representative bioluminescence images oftumor-bearing MDA-MB-231-D3H2LN mice treated with MN-RGD-anti-miR-10b(11B) or a control MN-scr-miR (11A) for four weeks beginning prior tolymph node metastasis.

FIG. 12 is a graph of the bioluminescence of lymph node tissue intumor-bearing MDA-MB-231-D3H2LN mice treated with MN-RGD-anti-miR-10b ora control MN-scr-miR for four weeks beginning prior to lymph nodemetastasis. The data shown are the mean±standard error (p≤0.01, n=3).

FIGS. 13A-D are a set of four representative bioluminescence images oftumor-bearing MDA-MB-231-D3H2LN mice treated with MN-RGD-anti-miR-10bfor four weeks beginning subsequent to lymph node metastasis. The micewere either treated with MN-anti-miR10b (13C and 13D) or controlMN-scr-miR (13A and 13D). Images taken at 1 week subsequent to lymphnode metastasis (top panels) and 4 weeks subsequent to lymph nodemetastasis (bottom panels) are shown.

FIG. 14 is a graph showing the fold change in the radiance of brachiallymph nodes from tumor-bearing MDA-MB-231-D3H2LN mice treated with theMN-anti-miR10b compared to the radiance of brachial lymph nodes fromtumor-bearing mice treated with MN-scr-miR at different time pointssubsequent to lymph node metastasis. The data shown are themean±standard error (p≤0.001, n=3).

FIGS. 15A and 15B are two immunofluorence images of lung tissue in thetumor-bearing MDA-MB-231-luc-D3H2LN mouse model treated withMN-anti-miR10b (15B) or MN-scr-miR (15A) beginning subsequent to lymphnode metastasis. Light shading indicates the presence of lung metastasesin the MDA-MB-231-luc-DH2LN mouse model.

FIG. 16 is a graph of flow cytometry data of 4T1 mouse metastatic breastcancer cells following a 24-hour incubation with MN-RGD-anti-miR-10b.

FIG. 17 a graph showing levels of miR-10b expression determined byquantitative reverse transcriptase polymerase chain reaction (qRT-PCR)in 4T1 mouse metastatic breast cancer cells following a 24-hourincubation with MN-RGD-anti-miR-10b or a corresponding magneticnanoparticle (MN-scr-miR) containing a scrambled nucleic acid ratherthan the anti-miR10b nucleic acid. The data shown are represented asmean±standard deviation (p≤0.0001, n=3).

FIG. 18 is a graph showing the effect on relative MDA-MB-231-D3H2LN inmice treated with the MN-anti-miR10b compared to the radiance ofbrachial lymph nodes from tumor-bearing mice treated with MN-scr-miR atdifferent time points subsequent to lymph node metastasis. There was nosignificant difference between experimental and control animals,incicating a lack of miR-10b influence on primary tumor growth (n=6).

FIG. 19 is a graph showing the tumor size of MDA-MB-231-D3H2LN tumorsexcised from mice treated with MN-anti-miR10b compared toMDA-MB-231-D3H2LN tumors excised form mice treated MN-scr-miR.

FIG. 20 is a graph showing the tumor weight of MDA-MB-231-D3H2LN tumorsexcised from mice treated with MN-anti-miR10b compared toMDA-MB-231-D3H2LN tumors excised form mice treated MN-scr-miR.

FIG. 21 is a set of Western blots showing an increase in HOXD10expression levels in MDA-MB-231-D3H2LN tumors and the lymph nodes withmetastatic burden after systematic MN-anti-miR10b administration.

FIG. 22 is a graph showing the lymph node metastatic burden followingtreatment with MN-anti-miR10b in the absence of a primary tumor. Thedata shown are the mean±standard error (n=6).

DETAILED DESCRIPTION

The therapeutic nanoparticles described herein were discovered todecrease cancer cell invasion and cancer metastasis in a mammal.Therapeutic nanoparticles having these activities are provided herein aswell as methods of decreasing cancer cell invasion or metastasis in asubject and methods of treating a metastatic cancer in a lymph node in asubject by administering these therapeutic nanoparticles.

Compositions

Provided herein are therapeutic nanoparticles that have a diameter ofbetween about 2 nm to about 200 nm (e.g., between about 10 nm to about30 nm, between about 5 nm to about 25 nm, between about 10 nm to about25 nm, between about 15 nm to about 25 nm, between about 20 nm and about25 nm, between about 25 nm to about 50 nm, between about 50 nm and about200 nm, between about 70 nm and about 200 nm, between about 80 nm andabout 200 nm, between about 100 nm and about 200 nm, between about 140nm to about 200 nm, and between about 150 nm to about 200 nm), andcontain a polymer coating, and a nucleic acid containing at least 10(e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21)contiguous nucleotides within the sequence of of GTGTAACACGTCTATACGCCCA(SEQ ID NO: 17), ATGGGACATCTTGGCTTAAACAC (SEQ ID NO: 1) orTGTCTAAGCTAAGAT CCCCTTA (SEQ ID NO: 2) that is covalently linked to thenanoparticle. In some embodiments, the therapeutic nanoparticles cancontain a sequence that is complementary to at least 10 (e.g., at least11,12,13,14,15,16,17,18,19,20,21,22, or 23) contiguous nucleotideswithin the sequence of precursor miR-I0b (e.g.,CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUGUAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCACAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUGC AAAAACUUCA; SEQ IDNO: 20). Mature and precursor miR-I0b are also described in WO07/073737, U.S. Patent Application Publication No. 2011/0107440, Ma etal. (Breast Cancer Res. 12:210,2010), Li et al. (Cancer Lett.299:29-36,2010), Ma et al. (Nat. Biotech. 28:341-347,2010), and Ma etal. 25 (Nature 449:682-688, 2007) (each of which is incorporated byreference in its entirety).

Alternatively, the nucleic acid contained in the therapeuticnanoparticles can contain a sequence that is complementary to a sequenceof at least 10 (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23) contiguous nucleotides present with a nucleic acidencoding an anti-apoptotic protein selected from the group of: survivin,XIAP, BCL2, BCL-XL, Mcl-1, Bfl-1, Bcl-W, Bcl-B, BRE, SGK1, MKP1/DUSP1,c-FLIP, MCL-1, MMP-15, BAG3, BIRC2, TRAP1, SCC-S2, HSP27, Livin, B7-H1,AAC-11, REG-1α, and HAX1. Alternatively, the nucleic acid contained inthe therapeutic nanoparticles can contain a sequence that iscomplementary to a sequence of at least 10 (e.g., at least 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotidespresent within mature human miR-21 or miR-125b.

In some embodiments, the therapeutic nanoparticles provided herein canbe spherical or ellipsoidal, or can have an amorphous shape. In someembodiments, the therapeutic nanoparticles provided herein can have adiameter (between any two points on the exterior surface of thetherapeutic nanoparticle) of between about 2 nm to about 200 nm (e.g.,between about 10 nm to about 200 nm, between about 2 nm to about 30 nm,between about 5 nm to about 25 nm, between about 10 nm to about 25 nm,between about 15 nm to about 25 nm, between about 20 nm to about 25 nm,between about 50 nm to about 200 nm, between about 70 nm to about 200nm, between about 80 nm to about 200 nm, between about 100 nm to about200 nm, between about 140 nm to about 200 nm, and between about 150 nmto about 200 nm). In some embodiments, therapeutic nanoparticles havinga diameter of between about 2 nm to about 30 nm localize to the lymphnodes in a subject. In some embodiments, therapeutic nanoparticleshaving a diameter of between about 40 nm to about 200 nm localize to theliver.

In some embodiments, the compositions can contain a mixture of two ormore of the different therapeutic nanoparticles described herein. Insome embodiments, the compositions contain at least one therapeuticnanoparticle containing at least 10 contiguous nucleotides within thesequence of SEQ ID NO: 1 or SEQ ID NO: 2 covalently linked to thenanoparticle (a nanoparticle for decrease miR-10b levels in a targetcell), and at least one therapeutic nanoparticle containing a sequencethat is complementary to a sequence of at least 10 contiguousnucleotides present within any one of SEQ ID NOS: 5-10 or within anucleic acid encoding an anti-apoptotic protein (e.g., any of theanti-apoptotic proteins described herein).

In some embodiments, the therapeutic nanoparticles can be magnetic(e.g., contain a core of a magnetic material).

Nanoparticles

In some embodiments, any of the therapeutic nanoparticles describedherein can contain a core of a magnetic material (e.g., a therapeuticmagnetic nanoparticle). In some embodiments, the magnetic material orparticle can contain a diamagnetic, paramagnetic, superparamagnetic, orferromagnetic material that is responsive to a magnetic field.Non-limiting examples of therapeutic magnetic nanoparticles contain acore of a magnetic material containing a metal oxide selected from thegroup of: magnetite; ferrites (e.g., ferrites of manganese, cobalt, andnickel); Fe(II) oxides, and hematite, and metal alloys thereof. The coreof magnetic material can be formed by converting metal salts to metaloxides using methods known in the art (e.g., Kieslich et al., Inorg.Chem. 2011). In some embodiments, the nanoparticles contain cyclodextringold or quantum dots. Non-limiting examples of methods that can be usedto generate therapeutic magnetic nanoparticles are described in Medarovaet al., Methods Mol. Biol. 555:1-13, 2009; and Medarova et al., NatureProtocols 1:429-431, 2006. Additional magnetic materials and methods ofmaking magnetic materials are known in the art. In some embodiments ofthe methods described herein, the position or localization oftherapeutic magnetic nanoparticles can be imaged in a subject (e.g.,imaged in a subject following the administration of one or more doses ofa therapeutic magnetic nanoparticle).

In some embodiments, the therapeutic nanoparticles described herein donot contain a magnetic material. In some embodiments, a therapeuticnanoparticle can contain, in part, a core of containing a polymer (e.g.,poly(lactic-co-glycolic acid)). Skilled practitioners will appreciatedthat any number of art known materials can be used to preparenanoparticles, including, but are not limited to, gums (e.g., Acacia,Guar), chitosan, gelatin, sodium alginate, and albumin. Additionalpolymers that can be used to generate the therapeutic nanoparticlesdescribed herein are known in the art. For example, polymers that can beused to generate the therapeutic nanoparticles include, but are notlimited to, cellulosics, poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactides(PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA),polyanhydrides, polyorthoesters, polycyanoacrylate and polycaprolactone.

Skilled practitioners will appreciate that the material used in thecomposition of the nanoparticles, the methods for preparing, coating,and methods for controlling the size of the nanoparticles can varysubstantially. However, these methods are well known to those in theart. Key issues include the biodegradability, toxicity profile, andpharmacokinetics/pharmacodynamics of the nanoparticles. The compositionand/or size of the nanoparticles are key determinants of theirbiological fate. For example, larger nanoparticles are typically takenup and degraded by the liver, whereas smaller nanoparticles (<30 nm indiameter) typically circulate for a long time (sometimes over 24-hrblood half-life in humans) and accumulate in lymph nodes and theinterstitium of organs with hyperpermeable vasculature, such as tumors.

Polymer Coatings

The therapeutic nanoparticles described herein contain a polymer coatingover the core magnetic material (e.g., over the surface of a magneticmaterial). The polymer material can be suitable for attaching orcoupling one or more biological agents (e.g., such as any of the nucleicacids, fluorophores, or targeting peptides described herein). One ofmore biological agents (e.g., a nucleic acid, fluorophore, or targetingpeptide) can be fixed to the polymer coating by chemical coupling(covalent bonds).

In some embodiments, the therapeutic nanoparticles are formed by amethod that includes coating the core of magnetic material with apolymer that is relatively stable in water. In some embodiments, thetherapeutic nanoparticles are formed by a method that includes coating amagnetic material with a polymer or absorbing the magnetic material intoa thermoplastic polymer resin having reducing groups thereon. A coatingcan also be applied to a magnetic material using the methods describedin U.S. Pat. Nos. 5,834,121, 5,395,688, 5,356,713, 5,318,797, 5,283,079,5,232,789, 5,091,206, 4,965,007, 4,774,265, 4,770,183, 4,654,267,4,554,088, 4,490,436, 4,336,173, and 4,421,660; and WO 10/111066 (eachdisclosure of which is incorporated herein by reference).

Method for the synthesis of iron oxide nanoparticles include, forexample, physical and chemical methods. For example, iron oxides can beprepared by co-precipitation of Fe2+ and Fe3+ salts in an aqueoussolution. The resulting core consists of magnetite (Fe₃O₄), maghemite(γ-Fe₂O₃) or a mixture of the two. The anionic salt content (chlorides,nitrates, sulphates etc), the Fe2+ and Fe3+ ratio, pH and the ionicstrength in the aqueous solution all play a role in controlling thesize. It is important to prevent the oxidation of the synthesizednanoparticles and protect their magnetic properties by carrying out thereaction in an oxygen free environment under inert gas such as nitrogenor argon. The coating materials can be added during the co-precipitationprocess in order to prevent the agglomeration of the iron oxidenanoparticles into microparticles. Skilled practitioners willappreciated that any number of art known surface coating materials canbe used for stabilizing iron oxide nanoparticles, among which aresynthetic and natural polymers, such as, for example, polyethyleneglycol (PEG), dextran, polyvinylpyrrolidone (PVP), fatty acids,polypeptides, chitosin, gelatin.

For example, U.S. Pat. No. 4,421,660 note that polymer coated particlesof an inorganic material are conventionally prepared by (1) treating theinorganic solid with acid, a combination of acid and base, alcohol or apolymer solution; (2) dispersing an addition polymerizable monomer in anaqueous dispersion of a treated inorganic solid and (3) subjecting theresulting dispersion to emulsion polymerization conditions. (col. 1,lines 21-27) U.S. Pat. No. 4,421,660 also discloses a method for coatingan inorganic nanoparticles with a polymer, which comprises the steps of(1) emulsifying a hydrophobic, emulsion polymerizable monomer in anaqueous colloidal dispersion of discrete particles of an inorganic solidand (2) subjecting the resulting emulsion to emulsion polymerizationconditions to form a stable, fluid aqueous colloidal dispersion of theinorganic solid particles dispersed in a matrix of a water-insolublepolymer of the hydrophobic monomer (col. 1, lines 42-50).

Alternatively, polymer-coated magnetic material can be obtainedcommercially that meets the starting requirements of size. For example,commercially available ultrasmall superparamagnetic iron oxidenanoparticles include NC100150 Injection (Nycomed Amersham, AmershamHealth) and Ferumoxytol (AMAG Pharmaceuticals, Inc.).

Suitable polymers that can be used to coat the core of magnetic materialinclude without limitation: polystyrenes, polyacrylamides,polyetherurethanes, polysulfones, fluorinated or chlorinated polymerssuch as polyvinyl chloride, polyethylenes, and polypropylenes,polycarbonates, and polyesters. Additional examples of polymers that canbe used to coat the core of magnetic material include polyolefins, suchas polybutadiene, polydichlorobutadiene, polyisoprene, polychloroprene,polyvinylidene halides, polyvinylidene carbonate, and polyfluorinatedethylenes. A number of copolymers, including styrene/butadiene,alpha-methyl styrene/dimethyl siloxane, or other polysiloxanes can alsobe used to coat the core of magnetic material (e.g., polydimethylsiloxane, polyphenylmethyl siloxane, and polytrifluoropropylmethylsiloxane). Additional polymers that can be used to coat the core ofmagnetic material include polyacrylonitriles or acrylonitrile-containingpolymers, such as poly alpha-acrylanitrile copolymers, alkyd orterpenoid resins, and polyalkylene polysulfonates. In some embodiments,the polymer coating is dextran.

Nucleic Acids

The therapeutic nanoparticles provided contain at least one nucleic acidcontaining at least 10 (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or 22) contiguous nucleotides within SEQ ID NO: 17, SEQ IDNO: 1 or SEQ ID NO: 2 that is covalently-linked to the nanoparticle. Insome embodiments, the nucleic acid contains the sequence of SEQ ID NO:17, SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acidcan contain a sequence of at least 10 (e.g., 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, or 23) contiguous nucleotides that is complementaryto a sequence within a nucleic acid encoding an anti-apoptotic proteinselected from the group of: survivin, XIAP, BCL2, BCL-XL, Mcl-1, Bfl-1,Bcl-W, Bcl-B, BRE, SGK1, MKP1/DUSP1, c-FLIP, MCL-1, MMP-15, BAG3, BIRC2,TRAP1, SCC-S2, HSP27, Livin, B7-H1, AAC-11, REG-1α, and HAX1. In someembodiments, the covalently-linked nucleic acid molecule contains asequence that is complementary to all or part of an mRNA encoding ananti-apoptotic protein (e.g., any of the anti-apoptotic proteinsdescribed herein). For example, the covalently-linked nucleic acid canbe complementary to all or part of a non-coding region of the codingstrand of a nucleotide sequence encoding an anti-apoptotic protein(e.g., any of the anti-apoptotic proteins described herein). Non-codingregions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequencesthat flank the coding region in a gene and are not translated into aminoacids. In some embodiments, the nucleic acid covalently-linked to thetherapeutic nanoparticle is complementary to the translational startcodon or a sequence encoding amino acids 1 to 5 of an anti-apoptoticprotein (e.g., any of the anti-apoptotic proteins described herein).

In some embodiments, the nucleic acid contained in the therapeuticnanoparticles can contain a sequence of at least 10 (e.g., at least 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) contiguousnucleotides within a sequence complementary to mature human miR-21 ormiR-125b. Mature human miR-125b has a sequence of ucccugagacccuaacuuguga (SEQ ID NO: 5), ucccugagacccuaacuuguga (SEQ ID NO: 6),ucccugagacccu aacuuguga (SEQ ID NO: 7), or ucacaagucaggcucuugggac (SEQID NO: 8). Mature human miR-21 has a sequence of cauugcacuugucucggucuga(SEQ ID NO: 9) or aggcggagacuugggcaauug (SEQ ID NO: 10).

The attached nucleic acid can be single-stranded or double-stranded. Insome embodiments, the nucleic acid contains the sequence of SEQ ID NO: 1and has a total length of between 23 nucleotides and 50 nucleotides(e.g., between 23-30 nucleotides, between 30-40 nucleotides, and between40-50 nucleotides). In some embodiments, the nucleic acid contains thesequence of SEQ ID NO: 2 and has a total length of between 22nucleotides and 50 nucleotides (e.g., between 22-30 nucleotides, between30-40 nucleotides, and between 40-50 nucleotides). In some embodiments,the nucleic acid can be an antisense RNA, a siRNA, or a ribozyme.

Antisense nucleic acid molecules can be covalently linked to thetherapeutic nanoparticles described herein. For example, nucleic acidsequences that contain a portion of the sequence (at least 10nucleotides) within SEQ ID NO: 1 are complementary to the sequence ofmature human miR-10b (uacccuguagaaccgaauuugug; SEQ ID NO: 3), andnucleic acid sequences that contain at least a portion of the sequence(e.g., at least 10 nucleotides) within SEQ ID NO: 2 are complementary tothe sequence of the minor form of mature human miR-10b(acagauucgauucuaggggaau; SEQ ID NO: 4). In some embodiments nucleic acidsequences that contain a portion of the sequence (at least 10nucleotides) within SEQ ID NO: 17 target human miR-10b fordown-regulation.

Based upon the sequences provided herein (e.g., the sequences for maturehuman miR-10b; SEQ ID NO: 3 and SEQ ID NO: 4; and the sequence precursormiR-10b; SEQ ID NO: 20), one of skill in the art can easily choose andsynthesize any of a number of appropriate antisense molecules (e.g.,antisense molecules to target mature human miR-10b). For example, anantisense nucleic acid that targets miR-10b can contain a sequencecomplementary to at least 10 (e.g., at least 15 or 20) contiguousnucleotides present in SEQ ID NO: 3 or SEQ ID NO: 4 or 20, or a sequencefor miR-10b known in the art.

An antisense nucleic acid can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides ormodified nucleotides (e.g., any of the modified oligonucleotidesdescribed herein) designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine-substituted nucleotides can be used.Alternatively, the antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., RNA transcribed from the insertednucleic acid will be of an antisense orientation to a target nucleicacid of interest). In some embodiments, the antisense nucleic acidmolecules described herein can hybridize to a target nucleic acid byconventional nucleotide complementarities and form a stable duplex.

An antisense nucleic acid molecule can be an α-anomeric nucleic acidmolecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gaultier et al.,Nucleic Acids Res. 15:6625-6641, 1987). The antisense nucleic acidmolecule can also comprise a 2′-O-methylribonucleotide (Inoue et al.,Nucleic Acids Res., 15:6131-6148, 1987) or a chimeric RNA-DNA analog(Inoue et al., FEBS Lett. 215:327-330, 1987).

In some embodiments, the nucleic acid is a ribozyme. For example, insome embodiments the nucleic acid is a ribozyme that has specificity formature human miR-10b in the cell (SEQ ID NO: 1 or 2). Ribozymes arecatalytic RNA molecules with ribonuclease activity that are capable ofcleaving a single-stranded nucleic acid, such as an RNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff et al. Nature 334:585-591, 1988)) can be used tocatalytically cleave RNA. A ribozyme having specificity for mature humanmiR-10b can be designed based upon the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 2. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved(complementary to SEQ ID NO: 1 or SEQ ID NO: 2, or any other contiguoussequence of at least 10 nucleotides in the miRNA-10b precursor) (U.S.Pat. Nos. 4,987,071 and 5,116,742). Alternatively, an oligonucleotidecontaining the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 17can be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel et al. Science261:1411-1418, 1993.

In some embodiments, the nucleic acid is a small interfering RNA(siRNA). RNAi is a process in which RNA is degraded in host cells. Todecrease expression of an RNA, double-stranded RNA (dsRNA) containing asequence corresponding to a portion of the target RNA (e.g., maturehuman miR-10b) is introduced into a cell. The dsRNA is digested into21-23 nucleotide-long duplexes called short interfering RNAs (orsiRNAs), which bind to a nuclease complex to form what is known as theRNA-induced silencing complex (or RISC). The RISC targets the endogenoustarget RNA by base pairing interactions between one of the siRNA strandsand the endogenous RNA. It then cleaves the endogenous RNA about 12nucleotides from the 3′ terminus of the siRNA (see Sharp et al., GenesDev. 15:485-490, 2001, and Hammond et al., Nature Rev. Gen. 2:110-119,2001).

Standard molecular biology techniques can be used to generate siRNAs.Short interfering RNAs can be chemically synthesized, recombinantlyproduced, e.g., by expressing RNA from a template DNA, such as aplasmid, or obtained from commercial vendors such as Dharmacon. The RNAused to mediate RNAi can include modified nucleotides (e.g., any of themodified nucleotides described herein), such as phosphorothioatenucleotides. The siRNA molecules used to decrease the levels of maturehuman miR-10b can vary in a number of ways. For example, they caninclude a 3′ hydroxyl group and strands of 21, 22, or 23 consecutivenucleotides. They can be blunt ended or include an overhanging end ateither the 3′ end, the 5′ end, or both ends. For example, at least onestrand of the RNA molecule can have a 3′ overhang from about 1 to about6 nucleotides (e.g., 1-5, 1-3, 2-4 or 3-5 nucleotides (whetherpyrimidine or purine nucleotides) in length. Where both strands includean overhang, the length of the overhangs may be the same or differentfor each strand.

To further enhance the stability of the RNA duplexes, the 3′ overhangscan be stabilized against degradation (by, e.g., including purinenucleotides, such as adenosine or guanosine nucleotides, or replacingpyrimidine nucleotides with modified nucleotides (e.g., substitution ofuridine two-nucleotide 3′ overhangs by 2′-deoxythymidine is toleratedand does not affect the efficiency of RNAi). Any siRNA can be usedprovided it has sufficient homology to the target of interest. There isno upper limit on the length of the siRNA that can be used (e.g., thesiRNA can range from about 21-50, 50-100, 100-250, 250-500, or 500-1000base pairs).

In some embodiments, the nucleic acid molecule can contain at least onemodified nucleotide (a nucleotide containing a modified base or sugar).In some embodiments, the nucleic acid molecule can contain at least onemodification in the phosphate (phosphodiester) backbone. Theintroduction of these modifications can increase the stability, orimprove the hybridization or solubility of the nucleic acid molecule.The molecules described herein can contain one or more (e.g., two,three, four, of five) modified nucleotides. The modified nucleotides cancontain a modified base or a modified sugar. Non-limiting examples ofmodified bases include: 8-oxo-N⁶-methyladenine, 7-deazaxanthine,7-deazaguanine, N⁴, N⁴-ethanocytosin, N⁶, N⁶-ethano-2,6-diaminopurine,5-(C³-C⁶)-alkynyl-cytosine, pseudoisocytosine,2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

Additional non-limiting examples of modified bases include thosenucleobases described in U.S. Pat. Nos. 5,432,272 and 3,687,808 (hereinincorporated by reference), Freier et al., Nucleic Acid Res.25:4429-4443, 1997; Sanghvi, Antisense Research and Application, Chapter15, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993; Englisch, et al.,Angewandte Chemie 30:613-722, 1991, Kroschwitz, Concise Encyclopedia ofPolymer Science and Engineering, John Wiley & Sons, pp. 858-859, 1990;and Cook, Anti-Cancer Drug Design 6:585-607, 1991. Additionalnon-limiting examples of modified bases include universal bases (e.g.,3-nitropyrole and 5-nitroindole). Other modified bases include pyreneand pyridyloxazole derivatives, pyrenyl, pyrenylmethylglycerolderivatives, and the like. Other preferred universal bases includepyrrole, diazole, or triazole derivatives, including those universalbases known in the art.

In some embodiments, the modified nucleotide can contain a modificationin its sugar moiety. Non-limiting examples of modified nucleotides thatcontain a modified sugar are locked nucleotides (LNAs). LNA monomers aredescribed in WO 99/14226 and U.S. Patent Application Publications Nos.20110076675, 20100286044, 20100279895, 20100267018, 20100261175,20100035968, 20090286753, 20090023594, 20080096191, 20030092905,20020128381, and 20020115080 (herein incorporated by reference).Additional non-limiting examples of LNAs are disclosed in U.S. Pat. Nos.6,043,060, 6,268,490, WO 01/07455, WO 01/00641, WO 98/39352, WO00/56746, WO 00/56748, and WO 00/66604 (herein incorporated byreference), as well as in Morita et al., Bioorg. Med. Chem. Lett.12(1):73-76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett.11(7):935-938, 2001; Koshkin et al., J. Org. Chem. 66(25):8504-8512,2001; Kvaerno et al., J. Org. Chem. 66(16):5498-5503, 2001; Hakansson etal., J. Org. Chem. 65(17):5161-5166, 2000; Kvaerno et al., J. Org. Chem.65(17):5167-5176, 2000; Pfundheller et al., Nucleosides Nucleotides18(9):2017-2030, 1999; and Kumar et al., Bioorg. Med. Chem. Lett.8(16):2219-2222, 1998. In some embodiments, the modified nucleotide isan oxy-LNA monomer, such as those described in WO 03/020739.

Modified nucleotides can also include antagomirs (2′-O-methyl-modified,cholesterol-conjugated single stranded RNA analogs); ALN (α-L-LNA); ADA(2′-N-adamantylmethylcarbonyl-2′-amino-LNA); PYR(2′-N-pyrenyl-1-methyl-2′-amino-LNA); OX (oxetane-LNA); ENA (2′-O,4″-C-ethylene bridged nucleic acid); AENA (2′-deoxy-2′-N,4′-C-ethylene-LNA); CLNA (2′,4′-carbocyclic-LNA); and CENA(2′,4′-carbocyclic-ENA); HM-modified DNAs (4′-C-hydroxymethyl-DNA);2′-substituted RNAs (with 2′-O-methyl, 2′-fluoro, 2′-aminoethoxymethyl,2′-aminopropoxymethyl, 2′-aminoethyl, 2′-guanidinoethyl, 2′-cyanoethyl,2′-aminopropyl); and RNAs with radical modifications of the ribose sugarring, such as Unlocked Nucleic Acid (UNA), Altritol Nucleic Acid (ANA)and Hexitol Nucleic Acid (HNA) (see, Bramsen et al., Nucleic Acids Res.37:2867-81, 2009).

The molecules described herein can also contain a modification in thephosphodiester backbone. For example, at least one linkage between anytwo contiguous (adjoining) nucleotides in the molecule can be connectedby a moiety containing 2 to 4 groups/atoms selected from the group of:—CH₂—, —O—, —S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO—,—S(O)₂—, —P(O)₂—, —PO(BH₃)—, P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—, and—PO(NHR^(H))—, where R^(H) is selected from hydrogen and C₁₋₄-alkyl, andR″ is selected from C₁₋₆-alkyl and phenyl. Illustrative examples of suchlinkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—, —O—CH₂—O—,—O—CH₂—CH₂—, —O—CH₂—CH═(including R⁵ when used as a linkage to asucceeding monomer), —CH₂—CH₂—O—, —NR^(H)—CH₂—CH₂—, —CH₂—CH₂—NR^(H)—,—CH₂—NR^(H)—CH₂—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—,—NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—, —NR^(H)—CO—CH₂—NR^(H)—,—O—CO—O—, —O—CO—CH₂—O—, —O—CH₂—CO—O—, —CH₂—CO—NR^(H)—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CH═N—O—,—CH₂—NR^(H)—O—, —CH₂—O—N═(including R⁵ when used as a linkage to asucceeding monomer), —CH₂—O—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—O—,—CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—, —O—NR^(H)—, —O—CH₂—S—, —S—CH₂—O—,—CH₂—CH₂—S—, —O—CH₂—CH₂—S—, —S—CH₂—CH═(including R⁵ when used as alinkage to a succeeding monomer), —S—CH₂—CH₂—, —S—CH₂—CH₂—O—,—S—CH₂—CH₂—S—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—, —O—SO—O—,—O—S(O)₂—O—, —O—S(O)₂—CH₂—, —O—S(O)₂—NR^(H)—, —NR_(H)—S(O)₂—CH₂—,—O—S(O)₂—CH₂—, —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O,S)—S—, —O—P(S)₂—S—, —S—P(O)₂—S—,—S—P(O,S)—S—, —S—P(S)₂—S—, —O—PO(R″)—O—, —O—PO(OCH₃)—O—,—O—PO—(OCH₂CH₃)—O—, —O—PO(OCH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(N))—O—,—O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))2-O—, —CH₂—P(O)₂—O—,—O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—; among which —CH₂—CO—NR^(H)—,—CH₂—NR^(H)—O—, —S—CH₂—O—, —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—,—NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and—O—PO(NHR^(N))—O—, where R^(H) is selected form hydrogen and C₁₋₄-alkyl,and R″ is selected from C₁₋₆-alkyl and phenyl. Further illustrativeexamples are given in Mesmaeker et. al., Curr. Opin. Struct. Biol.5:343-355, 1995; and Freier et al., Nucleic Acids Research 25:4429-43,1997. The left-hand side of the inter-nucleoside linkage is bound to the5-membered ring as substituent P* at the 3′-position, whereas theright-hand side is bound to the 5′-position of a preceding monomer.

In some embodiments, the deoxyribose phosphate backbone of the nucleicacid can be modified to generate peptide nucleic acids (see Hyrup etal., Bioorganic & Medicinal Chem. 4(1): 5-23, 1996). Peptide nucleicacids (PNAs) are nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs allows for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols, e.g.,as described in Hyrup et al., 1996, supra; Perry-O'Keefe et al., Proc.Natl. Acad. Sci. U.S.A. 93:14670-675, 1996.

PNAs can be modified, e.g., to enhance their stability or cellularuptake, by attaching lipophilic or other helper groups to PNA, by theformation of PNA-DNA chimeras, or by the use of liposomes or othertechniques of delivery known in the art. For example, PNA-DNA chimerascan be generated which may combine the advantageous properties of PNAand DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H, tointeract with the DNA portion while the PNA portion would provide highbinding affinity and specificity. PNA-DNA chimeras can be linked usinglinkers of appropriate lengths selected in terms of base stacking,number of bonds between the nucleobases, and orientation (Hyrup, 1996,supra). The synthesis of PNA-DNA chimeras can be performed as describedin Hyrup, 1996, supra, and Finn et al., Nucleic Acids Res. 24:3357-63,1996. For example, a DNA chain can be synthesized on a solid supportusing standard phosphoramidite coupling chemistry and modifiednucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., NucleicAcids Res., 17:5973-88, 1989). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., Nucleic Acids Res. 24:3357-63, 1996).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al., Bioorganic Med. Chem.Lett. 5:1119-11124, 1975).

In some embodiments, any of the nucleic acids described herein can bemodified at either the 3′ or 5′ end (depending on how the nucleic acidis covalently-linked to the therapeutic nanoparticle) by any type ofmodification known in the art. For example, either end may be cappedwith a protecting group, attached to a flexible linking group, orattached to a reactive group to aid in attachment to the substratesurface (the polymer coating). Non-limiting examples of 3′ or 5′blocking groups include: 2-amino-2-oxyethyl, 2-aminobenzoyl,4-aminobenzoyl, acetyl, acetyloxy, (acetylamino)methyl, 3-(9-acridinyl),tricyclo[3.3.1.1(3,7)]dec-1-yloxy, 2-aminoethyl, propenyl,(9-anthracenylmethoxy)carbonyl, (1,1-dmimethylpropoxy)carbonyl,(1,1-dimethylpropoxy)carbonyl,[1-methyl-1-[4-(phenylazo)phenyl]ethoxy]carbonyl, bromoacetyl,(benzoylamino)methyl, (2-bromoethoxy)carbonyl,(diphenylmethoxy)carbonyl, 1-methyl-3-oxo-3-phenyl-1-propenyl,(3-bromo-2-nitrophenyl)thio, (1,1-dimethyl ethoxy)carbonyl, [[(1,1-dimethylethoxy)carbonyl] amino]ethyl, 2-(phenylmethoxy)phenoxy,(1=[1,1′-biphenyl]-4-yl-1-methylethoxy) carbonyl, bromo,(4-bromophenyl)sulfonyl, 1H-benzotriazol-1-yl, [(phenylmethyl)thio]carbonyl, [(phenylmetyl)thio]methyl, 2-methylpropyl,1,1-dimethylethyl, benzoyl, diphenylmethyl, phenylmethyl, carboxyacetyl,aminocarbonyl, chlorodifluoroacetyl, trifluoromethyl,cyclohexylcarbonyl, cycloheptyl, cyclohexyl, cyclohexylacetyl, chloro,carboxymethyl, cyclopentylcarbonyl, cyclopentyl, cyclopropylmethyl,ethoxycarbonyl, ethyl, fluoro, formyl, 1-oxohexyl, iodo, methyl,2-methoxy-2-oxoethyl, nitro, azido, phenyl, 2-carboxybenzoyl,4-pyridinylmethyl, 2-piperidinyl, propyl, 1-methylethyl, sulfo, andethenyl. Additional examples of 5′ and 3′ blocking groups are known inthe art. In some embodiments, the 5′ or 3′ blocking groups preventnuclease degradation of the molecule.

The nucleic acids described herein can be synthesized using any methodsknown in the art for synthesizing nucleic acids (see, e.g., Usman etal., J. Am. Chem. Soc. 109:7845, 1987; Scaringe et al., Nucleic AcidRes. 18:5433, 1990; Wincott et al., Methods Mol. Biol. 74:59, 1997; andMilligan, Nucleic Acid Res. 21:8783, 1987). These typically make use ofcommon nucleic acid protecting and coupling groups. Synthesis can beperformed on commercial equipment designed for this purpose, e.g., a 394Applied Biosystems, Inc. synthesizer, using protocols supplied by themanufacturer. Additional methods for synthesizing the moleculesdescribed herein are known in the art. Alternatively, the nucleic acidscan be specially ordered from commercial vendors that synthesizeoligonucleotides.

In some embodiments, the nucleic acid is attached to the therapeuticnanoparticle at its 5′ end. In some embodiments, the nucleic acid isattached to the therapeutic nanoparticle at its 3′ end. In someembodiments, the nucleic acid is attached to the therapeuticnanoparticle through a base present in the nucleic acid.

In some embodiments, the nucleic acid (e.g., any of the nucleic acidsdescribed herein) is attached to the therapeutic nanoparticle (e.g., tothe polymer coating of the therapeutic nanoparticle) through a chemicalmoiety that contains a thioether bond or a disulfide bond. In someembodiments, the nucleic acid is attached to the therapeuticnanoparticle through a chemical moiety that contains an amide bond.Additional chemical moieties that can be used to covalently link anucleic acid to a therapeutic nanoparticle are known in the art.

A variety of different methods can be used to covalently link a nucleicacid to a therapeutic nanoparticle. Non-limiting examples of methodsthat can be used to link a nucleic acid to a magnetic particle aredescribed in EP 0937097; US RE41005; Lund et al., Nucleic Acid Res.16:10861, 1998; Todt et al., Methods Mol. Biol. 529:81-100, 2009; Brodyet al., J. Biotechnol. 74:5-13, 2000; Ghosh et al., Nucleic Acids Res.15:5353-5372, 1987; U.S. Pat. Nos. 5,900,481; 7,569,341; 6,995,248;6,818,394; 6,811,980; 5,900,481; and 4,818,681 (each of which isincorporated by reference in its entirety). In some embodiments,carboiimide is used for the end-attachment of a nucleic acid to atherapeutic nanoparticle. In some embodiments, the nucleic acid isattached to the therapeutic nanoparticle through the reaction of one ofits bases with an activated moiety present on the surface of thetherapeutic nanoparticle (e.g., the reaction of an electrophilic basewith a nucleophilic moiety on the surface of the therapeuticnanoparticle, or the reaction of a nucleophilic base with aelectrophilic residue on the surface of the therapeutic nanoparticle).In some embodiments, a 5′-NH₂ modified nucleic acid is attached to atherapeutic nanoparticle containing CNBr-activated hydroxyl groups (see,e.g., Lund et al., supra). Additional methods for attaching anamino-modified nucleic acid to a therapeutic nanoparticle are describedbelow. In some embodiments, a 5′-phosphate nucleic acid is attached to atherapeutic nanoparticle containing hydroxyl groups in the presence of acarbodiimide (see, e.g., Lund et al., supra). Other methods of attachinga nucleic acid to a therapeutic nanoparticle includecarboiimide-mediated attachment of a 5′-phosphate nucleic acid to a NH₂group on a therapeutic nanoparticle, and carboiimide-mediated attachmentof a 5′-NH₂ nucleic acid to a therapeutic nanoparticle having carboxylgroups (see, e.g., Lund et al., supra).

In exemplary methods, a nucleic acid can be produced that contains areactive amine or a reactive thiol group. The amine or thiol in thenucleic acid can be linked to another reactive group. The two commonstrategies to perform this reaction are to link the nucleic acid to asimilar reactive moiety (amine to amine or thiol to thiol), which iscalled homobifunctional linkage, or to link to the nucleic acid to anopposite group (amine to thiol or thiol to amine), known asheterobifunctional linkage. Both techniques can be used to attach anucleic acid to a therapeutic nanoparticle (see, for example, Misra etal., Bioorg. Med. Chem. Lett. 18:5217-5221, 2008; Mirsa et al., Anal.Biochem. 369:248-255, 2007; Mirsa et al., Bioorg. Med. Chem. Lett.17:3749-3753, 2007; and Choithani et al., Methods Mol Biol. 381:133-163,2007).

Traditional attachment techniques, especially for amine groups, haverelied upon homobifunctional linkages. One of the most common techniqueshas been the use of bisaldehydes such as glutaraldehyde. Disuccinimydylsuberate (DSS), commercialized by Syngene (Frederick, Md.) as syntheticnucleic acid probe (SNAP) technology, or the reagent p-phenylenediisothiocyanate can also be used to generate a covalent linkage betweenthe nucleic acid and the therapeutic nanoparticle.N,N′-o-phenylenedimaleimide can be used to cross-link thiol groups. Withall of the homobifunctional cross-linking agents, the nucleic acid isinitially activated and then added to the therapeutic nanoparticle (see,for example, Swami et al., Int. J. Pharm. 374:125-138, 2009, Todt etal., Methods Mol. Biol. 529:81-100, 2009; and Limanski{hacek over (i)},Biofizika 51:225-235, 2006).

Heterobifunctional linkers can also be used to attach a nucleic acid toa therapeutic nanoparticle. For example,N-succinidimidyl-3-(2-pyridyldithio)proprionate (SPDP) initially linksto a primary amine to give a dithiol-modified compound. This can thenreact with a thiol to exchange the pyridylthiol with the incoming thiol(see, for example, Nostrum et al., J. Control Release 15; 153(1):93-102,2011, and Berthold et al., Bioconjug. Chem. 21:1933-1938, 2010).

An alternative approach for thiol use has been a thiol-exchangereaction. If a thiolated nucleic acid is introduced onto a disulfidetherapeutic nanoparticle, a disulfide-exchange reaction can occur thatleads to the nucleic acid being covalently bonded to the therapeuticnanoparticle by a disulfide bond. A multitude of potential cross-linkingchemistries are available for the heterobifunctional cross-linking ofamines and thiols. Generally, these procedures have been used with athiolated nucleotide. Reagents typically employed have been NHS(N-hydroxysuccinimide ester), MBS (m-maleimidobenzoyl-N-succinimideester), and SPDP (a pyridyldisulfide-based system). Theheterobifunctional linkers commonly used rely upon an aminated nucleicacid. Additional methods for covalently linking a nucleic acid to atherapeutic nanoparticle are known in the art.

Targeting Peptides

The therapeutic nanoparticles described herein can also contain at leastone (e.g., two, three, or four) targeting peptide covalently-linked tothe therapeutic nanoparticle. Targeting peptides can be used to deliveran agent (e.g., any of the therapeutic nanoparticles described herein)to a specific cell type or tissue. Targeting peptides often contain anamino acid sequence that is recognized by a molecule present on thesurface of a cell (e.g., a cell type present in a target tissue). Forexample, a targeting peptide comprising an RGD peptide specificallybinds to aVf3.3 integrin expressed in the plasma membrane of breastcancer cells. Additional non-limiting targeting peptides that can becovalently-linked to any of the therapeutic nanoparticles describedherein include: an EPPT peptide, a contiguous sequence of amino acids(e.g., at least 10, 15, or 20) present within galectin-3, a contiguoussequence of amino acids (e.g., at least 10, 15, or 20) present withingonadotropin-releasing hormone, NYLHNHPYGTVG (SEQ ID NO: 11),SNPFSKPYGLTV (SEQ ID NO: 12), GLHESTFTQRRL (SEQ ID NO: 13), YPHYSLPGSSTL(SEQ ID NO: 14), SSLEPWHRTTSR (SEQ ID NO: 15), LPLALPRHNASV (SEQ ID NO:16), βAla-(Arg)₇-Cys (SEQ ID NO: 19) (e.g., C₁₄-βAla-(Arg)₇-Cys), acontiguous sequence of amino acids (e.g., at least 10, 15, or 20)present within somatostatin, a contiguous sequence of amino acids (e.g.,at least 10, 15, or 20) present within cholecystokinin-A, a contiguoussequence of amino acids (e.g., at least 10, 15, or 20) present withincholecystokinin-B, a contiguous sequence of amino acids (e.g., at least10, 15, or 20) present within glucagon-like peptide-1, a contiguoussequence of amino acids (e.g., at least 10, 15, or 20) present inbombesin, a contiguous sequence of amino acids (e.g., at least 10, 15,or 20) present within neuropeptide-Y, a contiguous sequence of aminoacids (e.g., at least 10, 15, or 20) present within vasoactiveintestinal peptide, a contiguous sequence of amino acids (e.g., at least10, 15, or 20) present within gastrin-1, a contiguous sequence of aminoacids (e.g., at least 10, 15, or 20) present within neurotensin, acontiguous sequence of amino acids (e.g., at least 10, 15, or 20)present within vascular endothelial growth factor, a contiguous sequenceof amino acids (e.g., at least 10, 15, or 20) present within endoglin,or a contiguous sequence of amino acids (e.g., at least 10, 15, or 20)present within epithelial growth factor. Additional examples oftargeting peptides are described in U.S. Patent Application PublicationNo. 2008/00056998 (herein incorporated by reference in its entirety).Additional examples of targeting peptides are known in the art.

In some embodiments, the targeting peptide can be covalently linked tothe therapeutic nanoparticle at its N-terminus or at its C-terminus. Insome embodiments, the targeting peptide can be covalently linked to thetherapeutic nanoparticle through an amino acid side chain.

Targeting peptides can be covalently-linked to any of the therapeuticnanoparticles described herein through a chemical moiety containing adisulfide bond, an amide bond, or a thioether bond. Additional chemicalmoieties that can be used to covalently link a targeting peptide to atherapeutic nanoparticle are known in the art.

A variety of different methods can be used to covalently link atargeting peptide to a therapeutic nanoparticle. Non-limiting examplesof methods of covalently linking a targeting peptide to a therapeuticnanoparticle are described in Hofmann et al., Proc. Nat. Acad. Sci.U.S.A. 10:3516-3518, 2007; Chan et al., PLoS ONE 2(11): e1164, 2007;U.S. Pat. No. 7,125,669; U.S. Patent Application Publication No.20080058224; U.S. Patent Application Publication No. 20090275066; andMateo et al., Nature Protocols 2:1022-1033, 2007 (each of which areincorporated by reference in their entirety). In some embodiments, thetherapeutic nanoparticle can be activated for attachment with atargeting peptide, for example in non-limiting embodiments, thetherapeutic nanoparticle can be epoxy-activated, carboxyl-activated,iodoacetyl-activated, aldehyde-terminated, amine-terminated, orthiol-activated. Additional methods for covalently linking a targetingpeptide to a therapeutic nanoparticle are known in the art.

Fluorophores

The therapeutic nanoparticles described herein can also contain at leastone (e.g., two, three, or four) fluorophore covalently-linked to thetherapeutic nanoparticle. In some embodiments, the fluorophore absorbsnear-infrared light (e.g., Cy5.5).

A variety of different fluorophores that can be covalently linked to thetherapeutic nanoparticles are known in the art. A variety of differentfluorophores that can be covalently linked to the therapeuticnanoparticles are known in the art. Non-limiting examples of suchfluorophores include: Sulforhodamine B, Resorufin,3,3-Diethylthiadicarbocyanine iodide, PdTFPP, Nile Red, DY-590, DY-590,Adams Apple Red 680, Adirondack Green 520, Birch Yellow 580, CatskillGreen 540, Fort Orange 600, Hemo Red, Hops Yellow, Lake Placid 490,Maple Red-Orange 620, Snake-Eye Red 900, QD525, QD565, QD585, QD605,QD655, QD705, QD800, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520,ATTO 550, ATTO 565, ATTO 590, ATTO 610, ATTO 620, ATTO 635, ATTO 647,ATTO 655, ATTO 680, ATTO 700, Alexa Fluor 633, 5-TAMRA, BOBO-3, Pro-QDiamond, resorufin, rhod-2, Rhodamine Red-X, rhodamine, R-phycoerythrin,sulforhodamine 101, tetramethylrhodamine, Texas Red-X, X-rhod-1, Calceinred-orange, Carboxynaphthofluorescein, DiIC18(3), Alexa Fluor 546, AlexaFluor 555, Alexa Fluor 610, Alexa Fluor 647, Alexa Fluor 680, JC-1,LOLO-1, tdTomato, mCherry, mPlum, mRaspberry, mRFP1.2, mStrawberry,mTangerine, CryptoLight CF4, CryptoLight CFS, CryptoLight CF6,R-phycoerythrin, SensiLight PBXL-3, Spectrum Orange, Spectrum Red,C3-Thiacyanine Dye, C5-Oxacyanine, nile red, MitoTracker Red CMXRos,MitoTracker Orange CMTMRos, LysoTracker Red DND-99, JC-1, Cy3.5,ReAsH-CCXXCC, AsRed2, DsRed, DsRed Dimer2, DsRed-Express T1,Fluorescein-Dibase, Magnesium Octaethylporphyrin, MagnesiumPhthalocyanine, Magnesium Phthalocyanine, Merocyanine 540,Phthalocyanine, Pinacyanol-Iodide, Rose Bengal basic, Sulforhodamine101, Tetra-t-Butylazaporphine, Zinc Octaethylporphyrin, ZincPhthalocyanine, Lumio Red, Rhodamine 700, Styryl 8 perchlorate,Terrylen, Terrylendiimid, Thionin acetate, Dye-304, Dye-1041, Dye-4,Cresyl Violet Perchlorate, DyLight 549, 2-dodecylresorufin, 5-ROX, AlexaFluor, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 594, PdOEPK,PtOEPK, Amplex UltraRed, BODIPY TR-X phallacidin, BODIPY TR-X, CalciumCrimson, CellTrace BODIPY TR methyl ester, CellTracker Red CMTPX, Cy3,DiI, FluoSpheres red, LDS 751, mRFP, pHrodo, succinimidyl ester, Qdot585, Qdot 605, QSY 7, QSY 9, Rhodamine phalloidin, Rhodamine Red-X,Tetramethylrhodamine, Texas Red, Texas Red DHPE, Lumogen Red F300Polystyren, Lumogen Red F300 Polystyren, Platinum(II)tetraphenyltetrabenzo-porphyrin, IRDye® 800CW Phosphate, and ATTO 647N.Additional non-limiting examples of near-infrared absorbing dyes arecommercially available from a variety of commercial vendors, includingQCR Solutions Corp. In some embodiments of the methods described herein,the position or localization of therapeutic nanoparticle containing acovalently-linked fluorophore can be imaged in a subject (e.g., imagedin a subject following the administration of one or more doses of atherapeutic nanoparticle containing a covalently-linked fluorophore).

In some embodiments, the fluorophore is attached to the therapeuticnanoparticle through a chemical moiety that contains a secondary amine,an amide, a thioester, or a disulfide bond. Additional chemical moietiesthat can be used to covalently link a fluorophore to a therapeuticnanoparticle are known in the art.

A variety of different methods can be used to covalently link afluorophore to a therapeutic nanoparticle. In some embodiments, thefluorophore is attached to the therapeutic nanoparticle through reactionof: an amino group (present in the fluorophore or on the therapeuticnanoparticle) with an active ester, carboxylate, isothiocyanate, orhydrazine (e.g., present in the fluorophore or on the therapeuticnanoparticle); through reaction of a carboxyl group (e.g., present inthe fluorophore or on the therapeutic nanoparticle) in the presence of acarbiimide; through reaction of a thiol (e.g., present in thefluorophore or on the therapeutic nanoparticle) in the presence ofmaleimide; through the reaction of a thiol (e.g., present in thefluorophore or on the therapeutic nanoparticle) in the presence ofmaleimide or aceyl bromide; or through the reaction of an azide (e.g.,present in the fluorophore or on the therapeutic nanoparticle) in thepresence of glutaraldehyde. Additional methods for attaching afluorophore to a therapeutic nanoparticle are known in the art.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions that contain atherapeutic nanoparticle as described herein. Two or more (e.g., two,three, or four) of any of the types of therapeutic nanoparticlesdescribed herein can be present in a pharmaceutical composition in anycombination. The pharmaceutical compositions can be formulated in anymanner known in the art.

Pharmaceutical compositions are formulated to be compatible with theirintended route of administration (e.g., intravenous, intraarterial,intramuscular, intradermal, subcutaneous, or intraperitoneal). Thecompositions can include a sterile diluent (e.g., sterile water orsaline), a fixed oil, polyethylene glycol, glycerine, propylene glycolor other synthetic solvents, antibacterial or antifungal agents such asbenzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like, antioxidants such as ascorbic acid or sodiumbisulfate, chelating agents such as ethylenediaminetetraacetic acid,buffers such as acetates, citrates, or phosphates, and isotonic agentssuch as sugars (e.g., dextrose), polyalcohols (e.g., manitol orsorbitol), or salts (e.g., sodium chloride), or any combination thereof.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers (see, e.g., U.S. Pat. No. 4,522,811). Preparations of thecompositions can be formulated and enclosed in ampules, disposablesyringes, or multiple dose vials. Where required (as in, for example,injectable formulations), proper fluidity can be maintained by, forexample, the use of a coating such as lecithin, or a surfactant.Absorption of the therapeutic nanoparticles can be prolonged byincluding an agent that delays absorption (e.g., aluminum monostearateand gelatin). Alternatively, controlled release can be achieved byimplants and microencapsulated delivery systems, which can includebiodegradable, biocompatible polymers (e.g., ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).Compositions containing one or more of any of the therapeuticnanoparticles described herein can be formulated for parenteral (e.g.,intravenous, intraarterial, intramuscular, intradermal, subcutaneous, orintraperitoneal) administration in dosage unit form (i.e., physicallydiscrete units containing a predetermined quantity of active compoundfor ease of administration and uniformity of dosage).

Toxicity and therapeutic efficacy of compositions can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals (e.g., monkeys). One can, for example, determine the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population): the therapeuticindex being the ratio of LD50:ED50. Agents that exhibit high therapeuticindices are preferred. Where an agent exhibits an undesirable sideeffect, care should be taken to minimize potential damage (i.e., reduceunwanted side effects). Toxicity and therapeutic efficacy can bedetermined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used informulating an appropriate dosage of any given agent for use in asubject (e.g., a human). A therapeutically effective amount of the oneor more (e.g., one, two, three, or four) therapeutic nanoparticles(e.g., any of the therapeutic nanoparticles described herein) will be anamount that treats decreases cancer cell invasion or metastasis in asubject having cancer (e.g., breast cancer) in a subject (e.g., ahuman), treats a metastatic cancer in a lymph node in a subject,decreases or stabilizes metastatic tumor size in a lymph node in asubject, decreases the rate of metastatic tumor growth in a lymph nodein a subject, decreases the severity, frequency, and/or duration of oneor more symptoms of a metastatic cancer in a lymph node in a subject ina subject (e.g., a human), or decreases the number of symptoms of ametastatic cancer in a lymph node in a subject (e.g., as compared to acontrol subject having the same disease but not receiving treatment or adifferent treatment, or the same subject prior to treatment).

The effectiveness and dosing of any of the therapeutic nanoparticlesdescribed herein can be determined by a health care professional usingmethods known in the art, as well as by the observation of one or moresymptoms of a metastatic cancer in a lymph node in a subject (e.g., ahuman). Certain factors may influence the dosage and timing required toeffectively treat a subject (e.g., the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and the presence of other diseases).

Exemplary doses include milligram or microgram amounts of any of thetherapeutic nanoparticles described herein per kilogram of the subject'sweight. While these doses cover a broad range, one of ordinary skill inthe art will understand that therapeutic agents, including thetherapeutic nanoparticles described herein, vary in their potency, andeffective amounts can be determined by methods known in the art.Typically, relatively low doses are administered at first, and theattending health care professional (in the case of therapeuticapplication) or a researcher (when still working at the developmentstage) can subsequently and gradually increase the dose until anappropriate response is obtained. In addition, it is understood that thespecific dose level for any particular subject will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, and the half-life of the therapeutic nanoparticles invivo.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The therapeutic nanoparticles described herein were discovered todecrease cancer cell invasion and to inhibit cancer cell metastasis. Inview of this discovery, provided herein are methods of decreasing cancercell invasion or metastasis in a subject, methods of treating ametastatic cancer in a lymph node in a subject, and methods ofdelivering a nucleic acid to a cell present in the lymph node of asubject. Specific embodiments and various aspects of these methods aredescribed below.

Methods of Treating Metastatic Cancer

Metastatic cancer is a cancer that originates from a cancer cell from aprimary tumor that has migrated to a different tissue in the subject. Insome embodiments, the cancer cell from the primary tumor can migrate toa different tissue in the subject by traveling through the blood streamor the lymphatic system of the subject. In some embodiments, themetastatic cancer is a metastatic cancer present in a lymph node in asubj ect.

The symptoms of metastatic cancer experienced by a subject depend on thesite of metastatic tumor formation. Non-limiting symptoms of metastaticcancer in the brain of a subject include: headaches, dizziness, andblurred vision. Non-limiting symptoms of metastatic cancer in the liverof a subject include: weight loss, fever, nausea, loss of appetite,abdominal pain, fluid in the abdomen (ascites), jaundice, and swellingof the legs. Non-limiting symptoms of metastatic cancer in the bone of asubject include: pain and bone breakage following minor or no injury.Non-limiting symptoms of metastatic cancer in the lung of a subjectinclude: non-productive cough, cough producing bloody sputum, chestpain, and shortness of breath.

A metastatic cancer can be diagnosed in a subject by a health careprofessional (e.g., a physician, a physician's assistant, a nurse, or alaboratory technician) using methods known in the art. For example, ametastatic cancer can be diagnosed in a subject, in part, by theobservation or detection of at least one symptom of a metastatic cancerin a subject (e.g., any of those symptoms listed above). A metastaticcancer can also be diagnosed in a subject using a variety of imagingtechniques (e.g., alone or in combination with the observance of one ormore symptoms of a metastatic cancer in a subject). For example, thepresence of a metastatic cancer (e.g., a metastatic cancer in a lymphnode) can be detected in a subject using computer tomography, magneticresonance imaging, positron emission tomography, and X-ray. A metastaticcancer (e.g., a metastatic cancer in a lymph node) can also be diagnosedby performing a biopsy of tissue from the subject (e.g., a biopsy of alymph node from the subject).

A metastatic tumor can form in a variety of different tissues in asubject, including, but not limited to: brain, lung, liver, bone,peritoneum, adrenal gland, skin, and muscle. The primary tumor can be ofany cancer type, including but not limited to: breast, colon, kidney,lung, skin, ovarian, pancreatic, rectal, stomach, thyroid, or uterinecancer.

Any one or more of the therapeutic nanoparticles described herein can beadministered to a subject having a metastatic cancer. The one or moretherapeutic nanoparticles can be administered to a subject in a healthcare facility (e.g., in a hospital or a clinic) or in an assisted carefacility. In some embodiments, the subject has been previously diagnosedas having a cancer (e.g., a primary cancer). In some embodiments, thesubject has been previously diagnosed as having a metastatic cancer(e.g., a metastatic cancer in the lymph node). In some embodiments, thesubject has already received therapeutic treatment for the primarycancer. In some embodiments, the primary tumor of the subject has beensurgically removed prior to treatment with one of the therapeuticnanoparticles described herein. In some embodiments, at least one lymphnode has been removed from the subject prior to treatment with one ofthe therapeutic nanoparticles described herein. In some embodiments, thesubject may be in a period of cancer remission.

In some embodiments, the administering of at least one therapeuticnanoparticle results in a decrease (e.g., a significant or observabledecrease) in the size of a metastatic tumor present in a lymph node, astabilization of the size (e.g., no significant or observable change insize) of a metastatic tumor present in a lymph node, or a decrease(e.g., a detectable or observable decrease) in the rate of the growth ofa metastatic tumor present in a lymph node in a subject. A health careprofessional can monitor the size and/or changes in the size of ametastatic tumor present in a lymph node in a subject using a variety ofdifferent imaging techniques, including but not limited to: computertomography, magnetic resonance imaging, positron emission tomography,and X-ray. For example, the size of a metastatic tumor present in alymph node of a subject can be determined before and after therapy inorder to determine whether there has been a decrease or stabilization inthe size of the metastatic tumor in the subject in response to therapy.The rate of growth of a metastatic tumor in the lymph node of a subjectcan be compared to the rate of growth of a metastatic tumor in anothersubject or population of subjects not receiving treatment or receiving adifferent treatment. A decrease in the rate of growth of a metastatictumor in the lymph node of a subject can also be determined by comparingthe rate of growth of a metastatic tumor in a lymph node both prior toand following a therapeutic treatment (e.g., treatment with any of thetherapeutic nanoparticles described herein). In some embodiments, thevisualization of a metastatic tumor (e.g., a metastatic tumor in a lymphnode) can be performed using imaging techniques that utilize a labeledprobe or molecule that binds specifically to the cancer cells in themetastatic tumor (e.g., a labeled antibody that selectively binds to anepitope present on the surface of the primary cancer cell).

In some embodiments, the administering of at least one therapeuticnanoparticle to the subject results in a decrease in the risk ofdeveloping an additional metastatic tumor in a subject already having atleast one metastatic tumor (e.g., a subject already having a metastatictumor in a lymph node) (e.g., as compared to the rate of developing anadditional metastatic tumor in a subject already having a similarmetastatic tumor but not receiving treatment or receiving an alternativetreatment). A decrease in the risk of developing an additionalmetastatic tumor in a subject already having at least one metastatictumor can also be compared to the risk of developing an additionalmetastatic tumor in a population of subjects receiving no therapy or analternative form of cancer therapy.

In some embodiments, administering a therapeutic nanoparticle to thesubject decreases the risk of developing a metastatic cancer (e.g., ametastatic cancer in a lymph node) in a subject having (e.g., diagnosedas having) a primary cancer (e.g., a primary breast cancer) (e.g., ascompared to the rate of developing a metastatic cancer in a subjecthaving a similar primary cancer but not receiving treatment or receivingan alternative treatment). A decrease in the risk of developing ametastatic tumor in a subject having a primary cancer can also becompared to the rate of metastatic cancer formation in a population ofsubjects receiving no therapy or an alternative form of cancer therapy.

A health care professional can also assess the effectiveness oftherapeutic treatment of a metastatic cancer (e.g., a metastatic cancerin a lymph node of a subject) by observing a decrease in the number ofsymptoms of metastatic cancer in the subject or by observing a decreasein the severity, frequency, and/or duration of one or more symptoms of ametastatic cancer in a subject. A variety of symptoms of a metastaticcancer are known in the art and are described herein. Non-limitingexamples of symptoms of metastatic cancer in a lymph node include: painin a lymph node, swelling in a lymph node, appetite loss, and weightloss.

In some embodiments, the administering can result in an increase (e.g.,a significant increase) the chance of survival of a primary cancer or ametastatic cancer in a subject (e.g., as compared to a population ofsubjects having a similar primary cancer or a similar metastatic cancerbut receiving a different therapeutic treatment or no therapeutictreatment). In some embodiments, the administering can result in animproved prognosis for a subject having a primary cancer or a metastaticcancer (e.g., as compared to a population of subjects having a similarprimary cancer or a similar metastatic cancer but receiving a differenttherapeutic treatment or no therapeutic treatment).

Methods of Decreasing Cancer Cell Invasion or Metastasis

Also provided are methods of decreasing (e.g., a significant orobservable decrease) cancer cell invasion or metastasis in a subjectthat include administering at least one therapeutic nanoparticledescribed herein to the subject in an amount sufficient to decreasecancer cell invasion or metastasis in a subject.

In some embodiments of these methods, the cancer cell metastasis is froma primary tumor (e.g., any of the primary tumors described herein) to asecondary tissue (e.g., a lymph node) in a subject. In some embodimentsof these methods, the cancer cell metastasis is from a lymph node to asecondary tissue (e.g., any of the secondary tissues described herein)in the subject.

In some embodiments, the cancer cell invasion is the migration of acancer cell into a tissue proximal to the primary tumor. In someembodiments, the cancer cell invasion is the migration of a cancer cellfrom a primary tumor into the lymphatic system. In some embodiments, thecancer cell invasion is the migration of a metastatic cancer cellpresent in the lymph node into the lymphatic system or the migration ofa metastatic cancer cell present in a secondary tissue to an adjacenttissue in the subject.

Cancer cell invasion in a subject can be assessed or monitored byvisualization using any of the imaging techniques described herein. Forexample, one or more tissues of a subject having a cancer or metastaticcancer can be visualized at two or more time points (e.g., at a timepoint shortly after diagnosis with a cancer and at later time point). Insome embodiments, a decrease in cancer cell invasion in a subject can bedetected by observing a decrease in the spread of a primary tumorthrough a specific tissue in the subject (when the spread of the primarytumor is assessed through the imaging techniques known in the art ordescribed herein). In some embodiments, a decrease in cancer cellinvasion can be detected by a reduction in the number of circulatingprimary cancer cells or circulating metastatic cancer cells in the bloodor lymph of a subject.

Cancer cell metastasis can be detected using any of the methodsdescribed herein or known in the art. For example, successful reductionof cancer cell metastasis can be observed as a decrease in the rate ofdevelopment of an additional metastatic tumor in a subject alreadyhaving at least one metastatic tumor (e.g., a subject already having ametastatic tumor in a lymph node) (e.g., as compared to the rate ofdevelopment of an additional metastatic tumor in a subject or apopulation of subjects already having a similar metastatic tumor but notreceiving treatment or receiving an alternative treatment). Successfulreduction of cancer cell metastasis can also be observed as a decreasein the risk of developing at least one metastatic cancer (e.g., ametastatic cancer in a lymph node) in a subject having (e.g., diagnosedas having) a primary cancer (e.g., a primary breast cancer) (e.g., ascompared to the risk of developing a metastatic cancer in a subject or apopulation of subjects having a similar primary cancer but not receivingtreatment or receiving an alternative treatment).

Dosing, Administration, and Compositions

In any of the methods described herein, the therapeutic nanoparticle canbe administered by a health care professional (e.g., a physician, aphysician's assistant, a nurse, or a laboratory or clinic worker), thesubject (i.e., self-administration), or a friend or family member of thesubject. The administering can be performed in a clinical setting (e.g.,at a clinic or a hospital), in an assisted living facility, or at apharmacy.

In some embodiments of any of the methods described herein, thetherapeutic nanoparticle is administered to a subject that has beendiagnosed as having a cancer (e.g., having a primary cancer or ametastatic cancer). In some embodiments, the subject has been diagnosedwith breast cancer (e.g., a metastatic breast cancer). In somenon-limiting embodiments, the subject is a man or a woman, an adult, anadolescent, or a child. The subject can have experienced one or moresymptoms of a cancer or metastatic cancer (e.g., a metastatic cancer ina lymph node). The subject can also be diagnosed as having a severe oran advanced stage of cancer (e.g., a primary or metastatic cancer). Insome embodiments, the subject may have been identified as having ametastatic tumor present in at least one lymph node. In someembodiments, the subject may have already undergone lymphectomy and/ormastectomy.

In some embodiments of any of the methods described herein, the subjectis administered at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, or 30) dose of a composition containing at least one (e.g.,one, two, three, or four) of any of the magnetic particles orpharmaceutical compositions described herein. In any of the methodsdescribed herein, the at least one magnetic particle or pharmaceuticalcomposition (e.g., any of the magnetic particles or pharmaceuticalcompositions described herein) can be administered intravenously,intaarterially, subcutaneously, intraperitoneally, or intramuscularly tothe subject. In some embodiments, the at least magnetic particle orpharmaceutical composition is directly administered (injected) into alymph node in a subject.

In some embodiments, the subject is administered at least onetherapeutic nanoparticle or pharmaceutical composition (e.g., any of thetherapeutic nanoparticles or pharmaceutical compositions describedherein) and at least one additional therapeutic agent. The at least oneadditional therapeutic agent can be a chemotherapeutic agent (e.g.,cyclophosphamide, mechlorethamine, chlorambucil, melphalan,daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone,valrubicin, paclitaxel, docetaxel, etoposide, teniposide, tafluposide,azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine,fluorouracil, gemcitabine, mercaptopurine, methotrexate, tioguanine,bleomycin, carboplatin, cisplatin, oxaliplatin, bortezomib, carfilzomib,salinosporamide A, all-trans retinoic acid, vinblastine, vincristine,vindesine, and vinorelbine) and/or an analgesic (e.g., acetaminophen,diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid,meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam,sulindac, tolmetin, celecoxib, buprenorphine, butorphanol, codeine,hydrocodone, hydromorphone, levorphanol, meperidine, methadone,morphine, nalbuphine, oxycodone, oxymorphone, pentazocine, propoxyphene,and tramadol).

In some embodiments, at least one additional therapeutic agent and atleast one therapeutic nanoparticle (e.g., any of the therapeuticnanoparticles described herein) are administered in the same composition(e.g., the same pharmaceutical composition). In some embodiments, the atleast one additional therapeutic agent and the at least one therapeuticnanoparticle are administered to the subject using different routes ofadministration (e.g., at least one additional therapeutic agentdelivered by oral administration and at least one therapeuticnanoparticle delivered by intravenous administration).

In any of the methods described herein, the at least one therapeuticnanoparticle or pharmaceutical composition (e.g., any of the therapeuticnanoparticles or pharmaceutical compositions described herein) and,optionally, at least one additional therapeutic agent can beadministered to the subject at least once a week (e.g., once a week,twice a week, three times a week, four times a week, once a day, twice aday, or three times a day). In some embodiments, at least two differenttherapeutic nanoparticles are administered in the same composition(e.g., a liquid composition). In some embodiments, at least onetherapeutic nanoparticle and at least one additional therapeutic agentare administered in the same composition (e.g., a liquid composition).In some embodiments, the at least one therapeutic nanoparticle and theat least one additional therapeutic agent are administered in twodifferent compositions (e.g., a liquid composition containing at leastone therapeutic nanoparticle and a solid oral composition containing atleast one additional therapeutic agent). In some embodiments, the atleast one additional therapeutic agent is administered as a pill,tablet, or capsule. In some embodiments, the at least one additionaltherapeutic agent is administered in a sustained-release oralformulation. In some embodiments, the one or more additional therapeuticagents can be administered to the subject prior to administering the atleast one therapeutic nanoparticle or pharmaceutical composition (e.g.,any of the therapeutic nanoparticles or pharmaceutical compositionsdescribed herein). In some embodiments, the one or more additionaltherapeutic agents can be administered to the subject afteradministering the at least one therapeutic nanoparticle orpharmaceutical composition (e.g., any of the magnetic particles orpharmaceutical compositions described herein). In some embodiments, theone or more additional therapeutic agents and the at least onetherapeutic nanoparticle or pharmaceutical composition (e.g., any of thetherapeutic nanoparticles or pharmaceutical compositions describedherein) are administered to the subject such that there is an overlap inthe bioactive period of the one or more additional therapeutic agentsand the at least one therapeutic nanoparticle (e.g., any of thetherapeutic nanoparticles described herein) in the subject.

In some embodiments, the subject can be administered the at least onetherapeutic nanoparticle or pharmaceutical composition (e.g., any of thetherapeutic nanoparticles or pharmaceutical compositions describedherein) over an extended period of time (e.g., over a period of at least1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years). Askilled medical professional may determine the length of the treatmentperiod using any of the methods described herein for diagnosing orfollowing the effectiveness of treatment (e.g., using the methods aboveand those known in the art). As described herein, a skilled medicalprofessional can also change the identity and number (e.g., increase ordecrease) of therapeutic nanoparticles (and/or one or more additionaltherapeutic agents) administered to the subject and can also adjust(e.g., increase or decrease) the dosage or frequency of administrationof at least one therapeutic nanoparticle (and/or one or more additionaltherapeutic agents) to the subject based on an assessment of theeffectiveness of the treatment (e.g., using any of the methods describedherein and known in the art). A skilled medical professional can furtherdetermine when to discontinue treatment (e.g., for example, when thesubject's symptoms are significantly decreased).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Uptake and Activity of Therapeutic Nanoparticles in BreastTumor Cells

Twenty to thirty-five nm dextran-coated therapeutic magneticnanoparticles were conjugated to the tumor-targeting peptide, cRGD.These therapeutic magnetic nanoparticles were further functionalizedwith knock-down locked nucleic acid (LNA) oligonucleotides (1.5 nmolesLNA/mL) targeting human miRNA-10b and the fluorophore, Cy5.5. Theresulting therapeutic magnetic nanoparticles are hereafter referred toas “MN-RGD-anti-miR-10b” (see, FIG. 1). These therapeutic magneticnanoparticles were generated as described below.

Synthesis of Therapeutic Magnetic Nanoparticles

Therapeutic magnetic nanoparticle (MN) synthesis was modified from aprotocol published previously (Medarova et al., Nat. Protocols1:429-435, 2006). Briefly, 9 g of Dextan-T10 (Pharmacosmos, Denmark) wasdissolved in 30 mL of double-distilled water and stirred in a roundbottom flask on ice. Iron (III) chloride hexahydrate (FeCl₃·6H₂O) (0.65g) was added while flushing Argon gas into the reaction mixture for anhour. Iron (II) chloride tetrahydrate (FeCl₂·4H₂O) (0.4 g) was addedinto the mixture, and then 15 mL of concentrated cold NH₄OH (˜28%) wasadded dropwise to the stirring mixture. The temperature increased to 85°C. for an hour to induce the formation of a nanoparticulate colloidalmixture, cooled to room temperature, and concentrated to 20 mL usingAmicon Ultra centrifugal units (molecular weight cut-off of 30 kDa;Millipore). The resulting 20 mL dextran-coated therapeutic magneticnanoparticles were cross-linked and aminated by the subsequent additionof 35 mL of 5 M NaOH, 14 mL of concentrated epichlorohydrin (8 hours)and 60 mL of concentrated NH₄OH. The nanoparticle solution was purifiedusing a dialysis bag (molecular weight cut-off of 14 kDa) against waterand 20 mM citrate buffer (pH 8.0), and then concentrated to 20 mL byAmicon Ultra centrifugal units. The nanoparticle concentration wasdetermined based on iron concentration (10.8 mg/mL Fe) and measuredspectrophotometrically, as described in Kumar et al. (Cancer Res.70:7553-7561, 2010). The size of the nanoparticles was determined bydynamic light scattering using Zetasizer Nano ZS (Malvern InstrumentsLtd) and nanoparticles of 23.5±3.5 nm were selected as suitable foraccumulation in tumors and lymph nodes. The number of amine groups wasfound to be 73 per nanoparticle.

One milligram of the near-infrared dye Cy5.5 monoreactive NHS ester (GEHealthcare) was dissolved in 100 μL of anhydrous DMSO and incubated withMN (10 mg Fe) in 20 mM citrate buffer (pH 8.0) overnight. Thenanoparticles were purified using Sephadex PD-10 column (GE Healthcare)against PBS. The number of Cy5.5 molecules per nanoparticle wasquantified spectrophotmometrically, and found to be four Cy5.5 moleculesper MN. The nanoparticles were further conjugated to heterofunctionallinker N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP) (PierceBiotechnology) in order to provide a thiol reactive terminus for LNA andcRGD conjugation. Briefly, 10 mg SPDP was dissolved in 500 μL anhydrousdimethyl sulfoxide (DMSO) and incubated with Cy5.5-labeled MN.

The MN were further conjugated to cRGD peptide through its cysteineterminus in PBS and purified with a Sephadex PD-10 column. The LNAoligonucleotides were then conjugated to MN. Briefly, the thiolated 5′terminus of the oligonucleotide was activated via 3% TCEP-treatment innuclease-free PBS. The LNA oligonucleotides were purified using ammoniumacetate/ethanol precipitation method. AfterTris(2-carboxyethyl)phosphine hydrochloride (TCEP)-activation andpurification, the oligonucleotides were resuspended in PBS and 50 mMethylenediaminetetraacetic acid (EDTA), and incubated with thenanoparticles overnight. The resulting probe was purified using a G-50Sephadex disposable quick spin columns (Roche Applied Science). Thequantification of cRGD and LNA per MN was performed as describedpreviously (Kumar et al., Cancer Res. 70:7553-7561, 2010) and determinedas fifteen cRGD per MN and ten LNA per MN.

Locked Nucleic Acids

The short locked nucleic acid (LNA) oligonucleotide sequence(anti-miR10b), 5′-ThioMC6-D/CACAAATTCGGTTCTACAGGGTA-3′ (SEQ ID NO: 18),directed against miRNA-10b, and a mismatch scrambled sequence (scr-miR),5′-Thio MC6-D/GTGTAACACGTCTATACGCCCA-3′ (SEQ ID NO: 17), weresynthesized by Exiqon Inc. A 5′-thiol modification was inserted intoboth sequences for conjugation to therapeutic magnetic nanoparticles anda 3′-Cy3 modification was inserted in the sequences for in vitrostudies. The thiol modifications on the oligonucleotides were activatedwith treatment of 3% Tris(2-carboxyethyl)phosphine hydrochloride,followed by purification with ammonium acetate/ethanol precipitation,prior to conjugation to the nanoparticles as described in Medarova etal. (Nat. Med. 13:372-377, 2007) and Kumar et al. (Cancer Res.70:7553-7561, 2010).

Synthesis of cRGD Peptide

The synthesis of Cyclic RGDfK-PEG-Cys-(Boc) was performed as follows.Cyclo Arg-Gly-Asp-D-Phe-Lys(PEG-PEG) (wherePEG=8-amino-3,6-dioxaoctanoic acid) (13.41 mg, 0.015 mmol, PeptideInternational Inc.) was added to a solution of Boc-Cys(Tris)-Osu (11.2mg, 0.02 mmol) in dimethylformamide (1 mL). The reaction mixture pH (pH8.5 to 9) was maintained using diisopropyl ethylmine, and the resultingreaction mixture was allowed to stir at room temperature overnight. Theproduct of the reaction was confirmed by thin layer chromatography, andisolated using the high pressure liquid chromatography (HPLC) gradientmethod. The collected fractions were lyophilized and obtained as a whitepowder. The final product was analyzed by MALDI-TOF mass spectrometry.

Cyclic RGDfK-PEG-Cys-(Boc) was treated with 2-3 mL of anhydroustrifluoroacetic acid at room temperature for 30 minutes. The resultingvolatile mixture was completely removed under vacuum. Afterwards, theresidue was dissolved in 100 mM NH₄OAc buffer (3 mL). The resultingsolution was filtered, and the filtrate was purified by HPLC. The finalproduct was analyzed by MALDI-TOF mass spectrometry.

Experiments were performed to determine whether MN-RGD-anti-miR-10bwould be taken up by human breast cancer cells. In these experiments,human breast cancer cells (MDA-MB-231 (gfp) cells) were incubated withMN-RGD-anti-miR-10b for 48 hours, and the data gathered using flowcytometry (for Cy5.5) and confocal microscopy (for Cy5.5 and LNA). Thedetails of the materials and methods used in these experiments aredescribed below.

Cell Lines

Human stably-transfected MDA-MB-231-luc-D3H2LN metastatic breast cancercell lines were authenticated based on viability, recovery, growth,morphology, and isoenzymology by the supplier (Caliper Life Sciences).The cells were passaged as recommended by the supplier.

Fluorescence Confocal Microscopy

Fluorescence confocal microscopy was performed on MDA-MB-231-GFP breastcancer cells. The cells (2×10⁶) were incubated with MN-anti-miR10b orMN-scr-miR (45 μg Fe, 4 nmols LNA) for 48 hours at 37° C. on a coverslip in an 8-well plate. The cells were then washed three times withHank's buffered salt solution (HBSS) and fixed with 2% formaldehyde for10 minutes. The cells were then washed three times with Dulbecco's PBS(DPBS), and the cover slip placed on a glass slide with Vectashieldmounting medium (Vector Laboratories). The slides were dried in a hoodfor 30 minutes in a dark room. The cells were imaged by confocalmicroscopy in the fluorescein isothiocyanate (FITC) channel (GFPdetection), the Cy3 channel (LNA detection), and the Cy5.5 channel (MNdetection) using a Zeiss LSM 5 Pascal laser confocal microscope. TheZeiss RGB vario laser module consists of an argon laser (458/488/514 nm)and two helium-neon lasers (543 and 633 nm). Image acquisition andanalyses were performed using Zeiss LSM 5 Pascal Confocal MicroscopySoftware (Release 3.2).

Flow Cytometry

MN-anti-miR10b and MN-scr-miR uptake by MDA-MB-231-GFP cells and 4T1 wasanalyzed by flow cytometry. MDA-MB-231-GFP cells were incubated with theprobes (45 μg Fe, 4 nmols LNA) for 48 hours and 4T1 cells were incubatedwith the probes (45 mg Fe, 4 nmol LNA) for 24 hours at 37° C. in an8-well plate, washed twice with HBBS buffer, and removed from the plateusing Hank's based enzyme-free cell dissociation buffer. The cells werethen fixed in 2% paraformaldehyde for 1 hour at 4° C. and diluted insheath solution for flow cytometry measurements. Nanodrug uptake wasanalyzed in the FL4 channel (Cy5.5, MN) and FL2 channel (Cy3, LNAoligonucleotides) using FACSCalibur (Becton Dickinson) equipped with theCellQuest software package.

The resulting data from these experiments show that 97.9% of theMDA-MB-231 cells took up MN-RGD-anti-miR-10b following 48 hours ofincubation (FIG. 2). Confocal microscopy of MDA-MB-231(gfp) cells alsoshowed staining for both Cy5.5 (blue) and LNA(red) following 48-hourincubation with MN-RGD-anti-miR-10b, further indicating the uptake ofMN-RGD-anti-miR-10b by these cells. The resulting data also show that74.6% of the 4T1 cells took up MN-RGD-anti-miR-10b following 24 hours ofincubation (FIG. 16).

Quantitative RT-PCR was performed to determine whether uptake ofMN-RGD-anti-miR-10b by the MDA-MB-231(gfp) cells results in a decreasein miR-10b levels in the cells. These experiments were performed asdescribed below.

Real-Time Quantitative Reverse Transcription-PCR

To measure the extent of miR-10b knockdown by the nanodrug, MDA-MB-231cells and 4T1 cells were incubated with MN-anti-miR10b and MN-scr-miR(45 μg 4 nmols LNA) for 48 hours at 37° C. The miRNA enriched fractionfrom total extracted RNA was harvested using the miRNeasy mini kit,according to the manufacturer's protocol (Qiagen, Inc.). The relativelevels of miR-10b were determined by real-time quantitative reversetranscription-PCR (qRT-PCR; Taqman protocol) and compared to theinternal housekeeping gene SNORD44. Taqman analysis was carried outusing an ABI Prism 7700 sequence detection system (PE AppliedBiosystems). The primers were provided by the manufacturer (RT2 miRNAFirst Strand Kit; SABiosciences).

The data from these experiments show that MDA-MB-231(gfp) cells treatedwith MN-RGD-anti-miR-10b for 48 hours have a 87.8±6.2% decrease inmiR-10b levels compared to MDA-MB-231(gfp) cells treated for 48 hourswith a corresponding magnetic nanoparticle containing a scramblednucleic acid rather than the anti-miR-10b nucleic acid (FIG. 3). Thesedata indicate that a ˜88% downregulation in miR-10b expression can beachieved by the administration of just 1.5 nmol/mL of LNA, whendelivered using MN-RGD-anti-miR-10b. The data from these experimentsalso show that MN-RGD-anti-miR-10b mediated a knock-down of the miR-10bexpression in 4T1 cells treated with MN-RGD-anti-miR-10b for 24 hourscompared to 4T1 cells treated for 24 hours with a corresponding magneticnanoparticle containing a scrambled nucleic acid rather than theanti-miR-10b nucleic acid (FIG. 17). Thus, the therapeutic nanoparticlesdescribed herein provide an efficient means for downregulating a targetnucleic acid (e.g., an miRNA involved in cancer cell invasion ormetastasis, or an anti-apoptotic mRNA) in a target cancer cell.

Additional experiments were performed in order to assess the toxicity ofMN-RGD-anti-miR-10b in MDA-MB-231(gfp) cells. In these experiments,MDA-MB-231(gfp) cells were treated with either MN-RGD-anti-miR-10b orcontrol magnetic nanoparticles (magnetic nanoparticles havinganti-miR-10b and no RGD; magnetic nanoparticles having controlLNA-scrambled nucleic acid and RGD; magnetic nanoparticles havingcontrol LNA-scrambled nucleic acid and no RGD) and apoptosis determinedusing a deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)assay. The resulting data show that treatment with MN-RGD-anti-miR-10bdoes not induce apoptotic cell death in MDA-MB-231(gfp) cells. Thesedata indicate a lack of MN-RGD-anti-miR-10b cytotoxicity.

Example 2. Therapeutic Nanoparticles Decrease Breast Tumor Cell Invasionand Migration

Additional experiments were performed to determine whether treatment ofbreast tumor cells with MN-RGD-anti-miR-10b would result in a decreasein tumor cell invasion and/or migration. In these experiments,MDA-MB-231(gfp) cells were treated with MN-RGD-anti-miR-10b or a controlmagnetic nanoparticle containing a scrambled oligonucleotide(MN-scr-miR) for 48 hours and then analyzed using standard cell invasionand migration kits (Cell Biolabs, Inc., San Diego, Calif.). Theseexperiments were performed according to the supplier's instructions.Briefly, MDA-MB-231 (0.1×106) cells were plated in each insertcontaining membrane (pore size 8 μm) and corresponding nanodrug (60 μg,5.0 nmol LNA) was added with/without 10% FCS (fetal calf serum), andincubated for 24 hours for the migration assay and 48 hours for theinvasion assay. The inserts were stained with the supplier's kit andmembranes are imaged by light microscopy to score migration andinvasion.

The resulting data show that migration and invasion stimulated by 10%FBS were both abrogated by MN-RGD-anti-miR-10b and not the controlmagnetic nanoparticles (MN-scr-miR) (see, FIGS. 4A-F and 5A-F). Thesedata indicate that administration of MN-RGD-anti-miR-10b to subjectscould have a significant impact on metastatic outcome in subjects havinga cancer.

Example 3. Therapeutic Nanoparticle Delivery to a Mouse Breast CancerModel

Additional experiments were performed in nude mice implantedorthotopically with the MDA-MB-231(luc) cell line to determine whetherthe therapeutic nanoparticles described herein would show significantlymphotropism when injected intravenously into tumor-bearing mice, andto determine whether the therapeutic nanoparticles described herein canbe used to treat primary and metastatic tumors in vivo. In vivo magneticresonance imagine (MM) was used to obtain information about the timecourse of magnetic particle delivery to tumor cells (Kumar et al.,Cancer Res. 7553-7561, 2010; Medarova et al., Cancer Res. 69:1182-1189,2009). These experiments were performed using the methods describedbelow.

Animal Models

Six-week old female nude mice (nu/nu or NIH III nude) were implantedorthotopically with the human breast adenocarcinomaMDA-MB-231-luc-D3H2LN cell line (Caliper Life Sciences, Hopkinton,Mass.). In this model, orthotopically-implanted tumors progress fromlocalized disease to lymph node metastasis. The tumor cells aretransformed with luciferase and can be detected by noninvasivebioluminescence imaging for correlative analysis of tumor burden.

In Vivo Magnetic Resonance Imaging (MRI)

MRI was performed before and 24-hours after intravenous administrationof the therapeutic magnetic nanoparticles using a 9.4T Bruker horizontalbore scanner with ParaVision 5.1 software. The imaging protocolconsisted of coronal T2-weighted spin echo (SE) pulse sequences with thefollowing parameters: SE repetition time/echo time (TE)=2000/[8, 16, 24,32, 40, 48, 56, 64]; field of view (FOV)=32×34 mm²; matrix size=128×128pixels; slice thickness=0.5 mm; in plane resolution=250×250 μm². Imageswere reconstructed and analyzed by Marevisi 3.5 software (Institute forBiodiagnostics, National Research Council, Canada). T2 maps wereconstructed according to established protocol by fitting T2 readings foreach of the eight TEs to a standard exponential decay curve.

T2 relaxation times were calculated by manually segmenting out the tumoron MR images from each slice for every animal before and after nanodruginjection. Longitudinal relaxation rate (R2=1/T2) was determined foreach slice and ΔR2 was calculated by subtracting the R2 readings beforefrom those after nanodrug administration.

The data from these experiments show that the therapeutic magneticnanoparticles are successfully delivered to the tumors, as shown by thedecrease in transverse relaxation time (T2; FIGS. 6A-B and 7).Quantitative analysis indicates a tendency towards MN-anti-miR10bbuild-up in the tissue after the second treatment session (FIG. 7).

Additional experiments were performed using near-infrared opticalimaging to study the whole-body distribution of MN-anti-miR10b inMDA-MB-231(luc) tumor-bearing mice. The MN-RGD-anti-miR-10b used inthese experiments was generated using a 23-nm magnetic nanoparticleprecursor as described in Example 1. The resulting therapeutic magneticnanoparticle, hereafter referred to as “23-nm MN-RGD-miR-10b”, wasinjected intravenously into the MDA-MB-231(luc) tumor-bearing mousemodel. Following injection, the tissue localization/bioavailability of23-nm MN-RGD-anti-miR-10b was assessed in the mice using in vivonear-infrared optimal imaging. These experiments were performed usingthe methods described below.

In Vivo Optical Imaging

In vivo fluorescence optical imaging was performed immediately aftereach in vivo MR imaging session. Anesthetized animals were placed supineinto a whole-body imaging system (IVIS Spectrum, Caliper Life Sciences)equipped with 10 narrow band excitation filters (30 nm bandwith) and 18narrow band emission filters (20 nm bandwith) that assist insignificantly reducing autofluorescence through spectral scanning offilters and the use of spectral unmixing algorithms (Caliper LifeSciences). The abdominal region of the animal was shielded in order toavoid interfering signal from internal organs. Imaging was performedusing 675-nm excitation and 720-nm emission filters. The epifluorescentimages and the grayscale photographs were acquired and superimposed.

The resulting data show that 23-nm MN-RGD-anti-miR-10b was taken up bythe primary tumor and lymph nodes (visible in the superficial axial,inguinal, and cervical lymph nodes) (FIGS. 8A-C). These data indicatethat 23-nm MN-RGD-anti-miR-10b represents a suitable approach fordelivery of an anti-miR-10b nucleic acid to both a primary tumor andlymph nodes (e.g., tumor cells present in the primary tumor and lymphnodes).

The accumulation of 23-nm MN-RGD-anti-miR-10b in the primary tumor andlymph nodes was also confirmed by ex vivo imaging. Ex vivo opticalimaging was performed as described below.

Ex Vivo Optical Imaging

Ex vivo fluorescent images were acquired by placing the excised tissuesin the imaging system immediately after the animals were sacrificed. Theaverage radiance from each fluorescence reading was used to estimatenanodrug uptake. The images were reconstructed using Living Imagesoftware version 4.0 (Caliper Life Sciences).

The resulting ex vivo imaging data show that 23-nm MN-RGD-anti-miR-10baccumulated in the primary tumor and brachial lymph nodes, an inguinallymph node, and a cervical lymph node in these tumor-bearing mice (FIGS.9A-E). Quantitative analysis also indicates a significantly high uptakeof the therapeutic magnetic nanoparticles by the primary tumor and lymphnodes relative to muscle tissue (FIG. 10).

Additional experiments were performed to determine the cellulardistribution of 23-nm MN-RGD-anti-miR-10b in tumor sections from thesemice. In these experiments, tumor sections from the tumor-bearing micetreated with 23-nm MN-RGD-anti-miR-10b were visualized usingnear-infrared fluorescence imaging (for Cy5.5). The resulting data showan extensive uptake of 23-nm MN-RGD-anti-miR-10b by the primary tumorpresent in these mice.

Experiments were also performed to determine whether a non-therapeuticmagnetic nanoparticle (MN-RGD-scrambled nucleic acid) would be taken upby metastatic tumor cells present in the lymph node of thesetumor-bearing mice. The MN-RGD-scrambled nucleic acid used in theseexperiments was generated by replacing the anti-miR-10b nucleic acidpresent in 20-nm MN-RGD-anti-miR-10b (derived from a 20-nm precursor)with a scrambled oligonucleotide. In these experiments, lymph nodesections were harvested from mice injected with MN-RGD-scrambled(MN-RGD-LNA(SCR)) and the distribution/localization of theMN-RGD-LNA(SCR) was assessed using staining for Cy5.5. Additionalco-staining was performed for CD68 (macrophages). These experiments wereperformed as described below.

Histology and Fluorescence Microscopy of Tissue Sections

To detect the metastatic lesions and/or the accumulation of thetherapeutic magnetic nanoparticles in a tissue (e.g., a primary tumor,lymph nodes, or lungs), the excised tissue was embedded in Tissue-TekOCT compound (Sakura Finetek) and snap frozen in liquid nitrogen. Thefrozen primary tumor and lymph nodes were cut into 7-μm sections. Thelungs were cut into 30-μm sections. The sections were fixed in 2%paraformaldehyde, washed, and counterstained with Vectashield mountingmedium containing 4′,6-diamidino-2-phenylindole (DAPI; VectorLaboratories), and analyzed by fluorescence microscopy using a NikonEclipse 50i fluorescence microscope equipped with the necessary filtersets. Images were acquired using a charge-coupled device camera withnear-IR sensitivity (SPOT 7.4 Slider RTKE; Diagnostic Instruments). Theimages were analyzed using SPOT 4.0 Advance version software (DiagnosticInstruments). The observed Cy5.5 signal within tumor sections was theresult of accumulation of the intravenously-injected magnetic particles.

For macrophage staining, frozen lymph node sections were incubated witha rat anti-mouse CD68 (FA-11) antibody (Serotec) followed by arhodamine-labeled goat secondary antibody (Abcam, Cambridge).Consecutive tissue sections were stained with hematoxylin and eosin(H&E), and analyzed by light microscopy to determine metastatic burdenor to compare fluorescent images with H&E-defined tissue architecture.

The resulting data show extensive uptake of MN-antimiR10b by the primarytumor cells and that MN-RGD-LNA(SCR) is taken up by both residentmacrophages in the lymph node and metastatic tumor cells located in thelymph node (metastatic tumor cells located in the paracortex outside ofthe lymphocyte-rich germinal centers). The data also show that in theabsence of metastatic tumor cells, the therapeutic nanoparticlesdistributed principally to macrophages. These data indicate that thetherapeutic nanoparticles described herein can target therapeuticnucleic acids to lymph nodes and can further target therapeutic nucleicacids to metastatic tumor cells present in lymph nodes.

Example 4. Therapeutic Nanoparticles Reduce Tumor Cell Metastasis InVivo

Additional experiments were performed to determine whetheradministration of MN-RGD-anti-miR-10b or MN-RGD-scrambled nucleic acid(control), starting prior to the beginning of tumor cell metastasis,would reduce tumor cell metastasis in the nude mouse model of humanbreast cancer (i.e., the orthotopic implantation of MDA-MB-231(luc)cells into nude mice). In vivo bioluminescence imaging was used tovisualize and quantify the metastatic burden from theluciferase-transformed MDA-MB-231-luc-D3H2L2 cell line. Theseexperiments were performed as described below.

Prevention of Metastasis

Six week-old nu/nu (n=12) were injected in the upper right mammary fatpad with 2×10⁶ MDA-MB-231-luc-D3H2LN cells (Caliper). The animals wereused in experiments 14-days after tumor implantation.

In Vivo Optical Bioluminescence Imaging

Mice were injected in the lower left abdominal quadrant with D-Luciferinpotassium salt firefly in DPBS (150 mg Luciferin/kg body weight, 200 μlof 15 mg/mL; Caliper Life Sciences) ten minutes before imageacquisition. The primary tumor was shielded to prevent signal leakageinto the right brachial lymph node. Identical imaging settings (time,30-60 seconds; F-stop, 2; binning, medium) and same-size regions ofinterest (ROIs) were used to obtain total radiance flux of themetastatic signals from right brachial lymph nodes. The total radiance(photons/second) from the bioluminescent readings was used for signalquantification.

The resulting data show that by the end of the treatment course, thesignal in experimental animals treated with MN-anti-miR10b was atpre-metastatic levels, indicating a prevention of tumor cell metastasisfrom primary tumor to lymph nodes (FIGS. 11A and B). In contrast, thedata show a visible dissemination of tumor cells to the lymph nodes ofcontrol animals treated with MN-scr-miR. This observed therapeuticeffect was highly reproducible (see FIG. 12). The therapeutic magneticnanoparticles in this experiment had a diameter of between 20-25 nm.

Example 5. Therapeutic Nanoparticles Arrest Metastasis

Additional experiments were performed to determine whether thetherapeutic magnetic nanoparticles could arrest metastatic disease onceit had spread beyond the primary tumor and into the lymph nodes. Inthese experiments, treatment with MN-anti-miR10b was initiated fourweeks after tumor cell implantation, subsequent to the formation oflymph node metastases. The methods used to perform these experiments aredescribed above with the modifications indicated below.

Arrest of Metastasis

Six week-old NIH III nude mice (n=6) were injected in the lower leftmammary fat pad with 2×10⁶ MDA-MB-231-luc-D3H2LN cells (Caliper). Theanimals were used in experiments 28-days after tumor implantation.Treatment with MN-anti-miR10b or MN-scr-miR involved systematicadministration through the tail vein at a dose of 10 mg Fe/kg once aweek, over four weeks.

In Vivo Optical Bioluminecense Imaging

For the study on metastatic arrest, the lower abdominal primary tumor inthe mammary fat was shielded and the total bioluminescence flux reading(photons/second) was taken from the right brachial lymph node with afixed ROI (F-stop, 8; binning large).

Fluorescence Microscopy

For luciferase staining, frozen lung sections were incubated with arabbit firefly luciferase antibody (Abcam, Cambridge, Mass.) followed bya DyLight® 488-labeled goat polyclonal secondary antibody to Rabbit IgG(Abcam, Cambridge, Mass.).

Western Blot

Frozen tumor sections were thawed and homogenized in tissue proteinextraction lysis buffer (Tissue-PE LB from G-Biosciences, St Louis, Mo.,USA) along with 1 mM phenylmethylsulfonyl fluoride (PMSF) and proteinaseinhibitor cocktails (Sigma, St Louis, Mo., USA). Protein content wasdetermined with the Bio-Rad protein assay kit (Bio-Rad, Hercules,Calif., USA). Lysates (50 mg) were separated by electrophoresis through4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis andtransferred to nitrocellulose membranes (Bio-Rad). Membranes wereblocked in 5% nonfat milk in Tris-buffered saline/Tween 20 for 1 hour atroom temperature.

After blocking, the membrane was incubated overnight at 41° C. in 1%milk/TBS containing HOXD10 (H-80) rabbit polyclonal antibody (1:200,Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and mouse monoclonalb-actin (1 mg/ml; Applied Biosystems, Carlsbad, Calif., USA). Themembrane was then washed three times with 0.05% Tween TBS (TBST) for 5minutes each and incubated with horseradish peroxidase-conjugated goatanti-rabbit or anti-mouse antibodies (Invitrogen, Camarillo, Calif.,USA) (1:2000 dilution) for 60 minutes at room temperature, followed bywashing three times with TBST and one time with TBS for 5 minutes each.Membranes were finally developed using ECL plus western blottingdetection reagents kit (GE Healthcare), according to the manufacturer'sspecifications.

The resulting data show that there was a complete arrest of metastasisin the experimental animals treated with MN-anti-miR10b (see FIGS. 13A-Dand 14). In contrast, mice treated with control MN-scr-miR had a 20-foldincrease in lymph node metastatic burden (FIG. 14), without aconcomitant effect on primary tumor growth (see FIGS. 18 and 19). Thiseffect was accompanied by induction of the known miR-10b target HOXD106(see FIG. 21) at both sites indicating that the function of miR-10b intumor cell migration is conserved between the primary and lymph nodemetastatic tumor cells.

The therapeutic effect of MN-anti-miR-10b also manifested as a decreasein distant metastasis. In the lungs of mice treated with MN-scr-miR,following the formation of metastasis in the lymph node, there weredistinct infiltrates of tumor cells; however, no tumor cells weredetected in the lungs of mice treated with MN-anti-miR10b, following theformation of metastasis in the lymph node (FIGS. 15A and 15B). Thesedata indicate that the magnetic particles described herein can arrestthe metastatic process and prevent further tumor cell colonization ofdistant organs (prevent or reduce further metastasis from a lymph nodeto a secondary tissue).

Example 6. Therapeutic Nanoparticles Arrest Metastasis in the Absence ofa Primary Tumor

Additional experiments were performed to determine whether thetherapeutic magnetic nanoparticles could arrest lymph node metastasis inthe absence of a primary tumor. In these experiments, the primary tumorswere surgically removed after establishment of lymph node metastasis andprior to the initiation of therapy. The methods used to perform theseexperiments are described above with the modifications indicated below.

Arrest of Metastasis in Mice with Surgically Removed Primary Tumor

Six-week-old nu/nu mice were injected in the lower left mammary fat padwith 2×106 MDA-MB-231-luc-D3H2LN cells (Caliper). Tumors were surgicallyremoved 28 days after tumor implantation. Treatment with MN-anti-miR10band MN-scr-miR involved systematic administration through the tail veinat a dose of 10 mg Fe/kg once a week over 4 weeks.

In Vivo Optical Bioluminecense Imaging

For the study on metastatic arrest, the lower abdominal primary tumor inthe mammary fat was shielded and the total bioluminescence flux reading(photons/second) was taken from the right brachial lymph node with afixed ROI (F-stop, 8; binning large).

The resulting data show there was an increase in bioluminescence signalfrom the brachial lymph nodes, consistent with metastatic expansion.(FIG. 22) By contrast, bioluminescence in the experimental animalsremained at pre-treatment levels, indicating that MN-anti-miR10b couldarrest the expansion of pre-established metastases. (FIG. 22)

Overall, the data described herein indicate that the therapeuticnanoparticles described herein can decrease tumor cell metastasis from aprimary tumor in a mammal (e.g., decrease tumor cell metastasis from aprimary tumor to a lymph node) and can further prevent or decrease tumorcell metastasis from the lymph node to a secondary tissue in a mammal.These data show that dextran-coated nanoparticles (e.g., 15-25 nm indiameter) are lymphotropic, with up to 9% of the injected doseaccumulating in lymph nodes. The increased uptake by lymph nodes isparticularly important when attempting to decrease or arrest metastaticprocess after the primary tumor cells have disseminated to lymph nodesin a mammal. The data further show that a significant fraction of theadministered therapeutic nanoparticles are taken up by macrophages, aswell as metastatic tumor cells, present in the lymph nodes.

Finally, these data suggest that the therapeutic nanoparticles providedherein can be used to target therapeutic nucleic acids to metastatictumor cells present in the lymph node, wherein the therapeutic nucleicacids result in a decrease or the stabilization of metastatic tumor sizeor result in a decrease in metastatic tumor growth.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A therapeutic nanoparticle, wherein saidtherapeutic nanoparticle: comprises a polymer coating, and a nucleicacid comprising at least 10 contiguous nucleotides within the sequenceof CACAAATTCGGTTCTACAGGGTA (SEQ ID NO: 18) that is covalently linked toa nanoparticle.
 2. The therapeutic nanoparticle of claim 1, wherein thenucleic acid comprises SEQ ID NO:
 18. 3. The therapeutic nanoparticle ofclaim 1, wherein the nucleic acid comprises at least one modifiednucleotide.
 4. The therapeutic nanoparticle of claim 3, wherein the atleast one modified nucleotide is a locked nucleotide.
 5. The therapeuticnanoparticle of claim 1, wherein the nanoparticle further comprises acovalently-linked fluorophore.
 6. The therapeutic nanoparticle of claim5, wherein the fluorophore absorbs near-infrared light.
 7. Thetherapeutic nanoparticle of claim 5, wherein the fluorophore iscovalently-linked to the nanoparticle through a chemical moietycomprising a secondary amine.
 8. The therapeutic nanoparticle of claim1, wherein the polymer coating comprises dextran.
 9. The therapeuticnanoparticle of claim 1, wherein the nucleic acid is a small interferingRNA (siRNA) molecule.
 10. The therapeutic nanoparticle of claim 1,wherein the nucleic acid is covalently-linked to the nanoparticlethrough a chemical moiety comprising a disulfide bond or a thioetherbond.
 11. The therapeutic nanoparticle of claim 1, wherein thenanoparticle is magnetic.
 12. A pharmaceutical composition comprisingthe therapeutic nanoparticle of claim 1 and a pharmaceuticallyacceptable carrier.
 13. A method for treating a metastatic cancer in asubject, the method comprising: administering to the subject in needthereof a therapeutic nanoparticle comprising a polymer coating and anucleic acid comprising at least 10 contiguous nucleotides within thesequence of CACAAATTCGGTTCTACAGGGTA (SEQ ID NO: 18) that is covalentlyor non-covalently linked to a nanoparticle, wherein the therapeuticnanoparticle is administered in an amount sufficient to treat themetastatic cancer in the subject.
 14. The method of claim 13, whereinthe therapeutic nanoparticle is administered to the subject byintravenous administration, subcutaneous, intraarterial, intramuscular,or intraperitoneal administration.
 15. The method of claim 13, whereinthe metastatic cancer is localized in breast, lymph nodes, lung, bone,brain, liver, ovary, peritoneum, muscle tissue, pancreas, prostate,esophagus, colon, rectum, stomach, nasopharyngeal or skin.
 16. Themethod of claim 13, wherein the nucleic acid comprises SEQ ID NO: 18.17. The method of claim 13, wherein the nucleic acid comprises at leastone modified nucleotide.
 18. The method of claim 17, wherein the atleast one modified nucleotide is a locked nucleotide.
 19. The method ofclaim 13, wherein the therapeutic nanoparticle further comprises acovalently-linked fluorophore.
 20. The method of claim 19, wherein thefluorophore absorbs near-infrared light.
 21. The method of claim 19,wherein the fluorophore is covalently-linked to the therapeuticnanoparticle through a chemical moiety comprising a secondary amine. 22.The method of claim 13, wherein the polymer coating comprises dextran.23. The method of claim 13, wherein the nucleic acid is covalentlylinked to the therapeutic nanoparticle through a chemical moietycomprising a disulfide bond or a thioether bond.
 24. The method of claim13, further comprising administering one or more additionalchemotherapeutic agents, wherein the one or more additionalchemotherapeutic agents is administered prior, concurrently, or afteradministration of the therapeutic nanoparticle.
 25. The method of claim24, wherein the one or more additional chemotherapeutic agents areselected from the group consisting of cyclophosphamide, mechlorethamine,chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide,teniposide, tafluposide, azacitidine, azathioprine, capecitabine,cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine,methotrexate, tioguanine, bleomycin, carboplatin, cisplatin,oxaliplatin, all-trans retinoic acid, vinblastine, vincristine,vindesine, vinorelbine, and bevacizumab.
 26. The method of claim 13,further comprising administering a radiotherapy treatment.
 27. Themethod of claim 13, further comprising administering molecularlytargeted therapy.
 28. The method of claim 27, wherein the molecularlytargeted therapy is Herceptin.